Method for sequencing a polynucleotide template

ABSTRACT

The invention relates to methods for pairwise sequencing of a double-stranded polynucleotide template, which methods result in the sequential determination of nucleotide sequences in two distinct and separate regions of the polynucleotide template. Using the methods of the invention it is possible to obtain two linked or paired reads of sequence information from each double-stranded template on a clustered array, rather than just a single sequencing read from one strand of the template.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. ProvisionalApplication No. 60/850,210, filed Oct. 6, 2006 and 60/898,910, filedFeb. 1, 2007. Applicants claim the benefits of priority under 35 U.S.C.§119 as to each of these provisional applications, and the entiredisclosure of each of these provisional applications is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods for pairwise sequencing of adouble-stranded polynucleotide template, which methods result in thesequential determination of nucleotide sequences in two distinct andseparate regions of the polynucleotide template.

BACKGROUND TO THE INVENTION

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of thesepublications and documents is incorporated by reference herein.

Advances in the study of biological molecules have been led, in part, byimprovement in technologies used to characterise the molecules or theirbiological reactions. In particular, the study of the nucleic acids DNAand RNA has benefited from developing technologies used for sequenceanalysis.

One method for sequencing a polynucleotide template involves performingmultiple extension reactions using a DNA polymerase to successivelyincorporate labelled nucleotides to a template strand. In such a“sequencing by synthesis” reaction a new nucleotide strand base-pairedto the template strand is built up in the 5′ to 3′ direction bysuccessive incorporation of individual nucleotides complementary to thetemplate strand. If used simultaneously, the substrate nucleosidetriphosphates used in the sequencing reaction may be blocked to preventover-incorporation and labelled differently, permitting determination ofthe identity of the incorporated nucleotide as successive nucleotidesare added.

In order to carry out accurate sequencing a reversible chain-terminatingstructural modification or “blocking group” may be added to thesubstrate nucleotides to ensure that nucleotides are incorporated one ata time in a controlled manner. As each single nucleotide isincorporated, the blocking group prevents any further nucleotideincorporation into the polynucleotide chain. Once the identity of thelast-incorporated labelled nucleotide has been determined the labelmoiety and blocking group are removed, allowing the next blocked,labelled nucleotide to be incorporated in a subsequent round ofsequencing.

In certain circumstances the amount of sequence data that can bereliably obtained with the use of sequencing-by-synthesis techniques,particularly when using blocked, labelled nucleotides, may be limited.In some circumstances it is preferred to limit the sequencing “run” to anumber of bases that permits sequence realignment with the human genome,typically around 25-30 cycles of incorporation. Whilst sequencing runsof this length are extremely useful, particularly in applications suchas, for example, SNP analysis and genotyping, it would be advantageousin many circumstances to be able to reliably obtain further sequencedata for the same template molecule.

The technique of “paired-end” or “pairwise” sequencing is generallyknown in the art of molecular biology, particularly in the context ofwhole-genomic shotgun sequencing. Paired-end sequencing allows thedetermination of two “reads” of sequence from two places on a singlepolynucleotide duplex. The advantage of the paired-end approach is thatthere is significantly more information to be gained from sequencing twostretches each of “n” bases from a single template than from sequencing“n” bases from each of two independent templates in a random fashion.With the use of appropriate software tools for the assembly of sequenceinformation it is possible to make use of the knowledge that the“paired-end” sequences are not completely random, but are known to occuron a single duplex, and are therefore linked or paired in the genome.This information has been shown to greatly aid the assembly of wholegenome sequences into a consensus sequence.

Paired-end sequencing has typically been performed by making use ofspecialized circular shotgun cloning vectors. After cutting the vectorat a specific single site, the template DNA to be sequenced (typicallygenomic DNA) is inserted into the vector and the ends resealed to form anew construct. The vector sequences flanking the insert DNA includebinding sites for sequencing primers which permit sequencing of theinsert DNA on opposite strands. However, the need for sequencing primersat both ends of the template fragment makes the use of array-basedsequencing techniques extremely difficult. With array-based techniques,which usually rely on a single stranded template, it is generally onlypossible to sequence from one end of a nucleotide template, as thecomplementary strand is not attached to the surface.

A number of methods for double-ended sequencing of a polynucleotidetemplate which can be carried out on a solid support have been reported,for example US20060024681, US20060292611, WO06110855, WO06135342,WO03074734, WO007010252, WO07091077 and WO00179553.

WO 98/44151 and WO 00/18957 both describe methods of nucleic acidamplification which allow amplification products to be immobilised on asolid support in order to form arrays comprised of clusters or“colonies” formed from a plurality of identical immobilisedpolynucleotide strands and a plurality of identical immobilisedcomplementary strands. The nucleic acid molecules present in DNAcolonies on the clustered arrays prepared according to these methods canprovide templates for sequencing reactions, for example as described inWO 98/44152. It is advantageous to enable the efficient sequencing ofboth strands of such clusters, as described in detail in the methodsherein.

SUMMARY OF THE INVENTION

The present inventors have now developed methods for paired-endsequencing of double-stranded polynucleotide templates, includingdouble-stranded templates present on clustered arrays, such as thosedescribed herein. The methods permit sequencing of two distinct regions,one at each end of the complementary strands of a target polynucleotideduplex. Using the methods of the invention it is possible to obtain twolinked or paired reads of sequence information from each double-strandedtemplate on a clustered array, rather than just a single sequencing readfrom one strand of the template.

According to one method of the invention there is provided a method forpairwise sequencing of first and second regions of a targetdouble-stranded polynucleotide, wherein said first and second regionsare in the same target double-stranded polynucleotide, the methodcomprising:

-   (a) providing a solid support having immobilised thereon a plurality    of double stranded template polynucleotides each formed from    complementary first and second template strands linked to the solid    support at their 5′ ends, and multiple copies of one or more 5′-end    immobilised primers capable of hybridising to the 3′ end of the    first template strand;-   (b) treating the plurality of double stranded template    polynucleotides such that the first template strands are hybridised    to 5′-end immobilised primers;-   (c) carrying out a first sequencing read to determine the sequence    of a first region of the template polynucleotide;-   (d) carrying out an extension reaction to extend one or more of the    immobilised primers to copy the first template strand to generate a    second immobilised template strand;-   (e) treating the plurality of first and second immobilised template    strands to remove the first template strand from the solid support;-   (f) carrying out a second sequencing read to determine the sequence    of a second region of the template polynucleotide, wherein    determining the sequences of the first and second regions of the    target polynucleotide achieves pairwise sequencing of said first and    second regions of said target double-stranded polynucleotide.

A second method of the invention provides a method for sequencing endregions A and B of a target double-stranded polynucleotide, wherein saidend regions A and B are in the same target double-strandedpolynucleotide, the method comprising:

-   (a) providing a solid support having immobilised thereon a plurality    of double stranded template polynucleotides each formed from    complementary first and second template strands linked to the solid    support at their 5′ ends;-   (b) treating the double stranded template polynucleotides such that    each double stranded template polynucleotide is cut in at least two    places to generate two shortened double stranded template fragments    A and B immobilised at one end, wherein A and B are no longer    directly connected;-   (c) treating the two shortened double stranded template fragments A    and B immobilised at one end to make the two non-immobilised ends a    blunt ended duplex;-   (d) treating the two blunt ended duplexes such that the two blunt    ends A and B are connected to form a double stranded nucleotide    sequence containing both ends A and B of the original target    fragment in a shortened contiguous sequence, immobilised at both    ends;-   (e) cleaving one strand of the double stranded nucleotide sequence    containing both distal ends A and B of the original target fragment    joined in a shortened contiguous sequence immobilised at both ends    to generate a single stranded nucleotide target sequence containing    both distal ends A and B of the original target fragment in a    shortened contiguous sequence, wherein said single stranded    nucleotide target sequence is immobilised at a single 5′ or 3′ end;-   (f) hybridising a sequencing primer to the single stranded    nucleotide target sequence containing both distal ends A and B of    the original target fragment in a shortened contiguous sequence; and-   (g) carrying out a single sequencing reaction to determine a    contiguous sequence of both ends A and B of the original target    fragment.

A third method of the invention provides for pairwise sequencing offirst and second regions of a target double-stranded polynucleotide,wherein said first and second regions are in the same targetdouble-stranded polynucleotide, the method comprising:

-   (a) providing a solid support having immobilised thereon a plurality    of double stranded template polynucleotides each formed from    complementary first and second template strands linked to the solid    support at both their 5′ ends, and multiple copies of one or more    5′-end immobilised primers capable of hybridising to the 3′ end of    the first template strand;-   (b) treating the plurality of double stranded template    polynucleotides such that the first template strands are hybridised    to a primer that is immobilised on the solid support at its 5′-end;-   (c) carrying out a first sequencing to determine the sequence of a    first region of the template polynucleotide;-   (d) carrying out an extension reaction to extend one or more of the    immobilised primers to the end of the first template strand to    generate a second immobilised template strand;-   (e) treating the plurality of template polynucleotides such that the    first template strand is removed from the solid support leaving the    second immobilised template strand single stranded;-   (f) carrying out a second sequencing read to determine the sequence    of a second region of the template polynucleotide, wherein    determining the sequences of the first and second regions of the    target polynucleotide achieves pairwise sequencing of said first and    second regions of said target double-stranded polynucleotide.

In a more specific example, said third method is a method for pairwisesequencing of first and second regions of a target double-strandedpolynucleotide, wherein said first and second regions are in the sametarget double-stranded polynucleotide, the method comprising:

-   (a) providing a solid support having immobilised thereon a plurality    of double stranded template polynucleotides each formed from    complementary first and second template strands linked to the solid    support at their 5′ ends;-   (b) treating the plurality of double stranded template    polynucleotides such that one of the strands is released from the    surface leaving a single stranded first template strand immobilised    on the solid support at its 5′-end;-   (c) hybridising a primer to said first template strand and carrying    out a first sequencing reaction to monitor the incorporation of    labelled nucleotides onto the hybridised primer using cycles of    primer extension with a polymerase and labelled nucleotides to    generate a first extended sequencing primer and determine the    sequence of a first region of the template polynucleotide;-   (d) removing said first extended sequencing primer;-   (e) hybridising the immobilised first template strand with    immobilised primers and extending said immobilised primers to    regenerate said plurality of double stranded template    polynucleotides each formed from complementary first and second    template strands linked to the solid support at their 5′ ends;-   (f) treating the plurality of template polynucleotides such that the    first template strand is removed from the surface leaving the second    immobilised template strand single stranded;-   (g) hybridising a second sequencing primer to the second immobilised    template strands; and-   (h) carrying out a second sequencing run to monitor the    incorporation of labelled nucleotides onto the second sequencing    primer using cycles of primer extension with a polymerase and    labelled nucleotides to generate a second extended sequencing primer    and determine the sequence of a second region of the template    polynucleotide, wherein determining the sequences of the first and    second regions of the target polynucleotide achieves pairwise    sequencing of said first and second regions of said target    double-stranded polynucleotide.

Further covered within the embodiments of the inventions are clusteredarrays prepared according to any method described herein, for exampleusing strand resynthesis between two sequencing reads, or using arestriction endonuclease treatment to excise the central region of animmobilised duplex.

Further described herein is a method of improving the data quality of asequencing reaction on an immobilised template, the method comprisinghybridising the template to an immobilised primer such that the templateis immobilised through both ends.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of a paired-end read using a first method ofthe invention enabled with a nicking enzyme.

FIG. 2 shows a schematic of a paired read using a first method of theinvention enabled with a uracil primer.

FIG. 3 shows a schematic of a paired read using a second method of theinvention.

FIG. 4 a shows a schematic of a paired read using a third method of theinvention. The figure shows the first sequencing read occurring from ahybridised primer, but the strand may also be hybridised to thephosphate blocked primer on the surface, and hence may be immobilisedvia both ends (as shown in FIG. 4 c).

FIG. 4 b shows a schematic of a paired read using the third method ofthe invention enabled using three grafting primers.

FIG. 4 c shows the method of FIG. 4 a, where the template is immobilisedthrough both ends during SBS read 1.

FIG. 5 shows a representation of the clusters as grown on the surface.

FIG. 6 shows the full sequence of one strand of the template used foramplification.

FIG. 7 shows data from two cycles of nucleotide incorporation ontoimmobilised sequencing primer.

FIG. 8 shows data from sequencing reads obtained from the method shownin FIG. 4 b. The sample used was a fragmented BAC of 140 KB fragmentedto an average insert size of 80 base pairs. The two reads were obtainedfrom either end of the fragment. Numerical data from a single tile of asequencing run is shown. Both reads clearly align against the BACsequence, with only 4% of the read derived from E. coli thatcontaminated the original sample.

FIG. 9 shows a schematic of a method using two nicking enzymes andstrand resynthesis.

FIG. 10 shows the optional step of primer extension prior to strandresynthesis, which improves the strand resynthesis step.

FIG. 11 shows an alternative method for reversibly blocking theimmobilised primer.

FIG. 12 shows a schematic of indexing paired reads, wherein the sampletemplates are prepared using an indexing sequence or tag.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for sequencing two regions of a targetdouble-stranded polynucleotide template, referred to herein as the firstand second regions for sequence determination. The first and secondregions for sequence determination are at both ends of complementarystrands of the double-stranded polynucleotide template, which arereferred to herein respectively as first and second template strands.Once the sequence of a strand is known, the sequence of itscomplementary strand is also known, therefore the term two regions canapply equally to both ends of a single stranded template, or both endsof a double stranded template, wherein a first region and its complementare known, and a second region and its complement are known.

The starting point for the method of the invention is the provision of aplurality of template polynucleotide duplexes immobilised on a solidsupport. The template polynucleotides may be immobilised in the form ofan array of amplified single template molecules, or ‘clusters’. Each ofthe duplexes within a particular cluster comprises the samedouble-stranded target region to be sequenced. The duplexes are eachformed from complementary first and second template strands which arelinked to the solid support at or near to their 5′ ends. Typically, thetemplate polynucleotide duplexes will be provided in the form of aclustered array.

An alternate starting point is a plurality of single stranded templateswhich are attached to the same surface as a plurality of primers thatare complementary to the 3′ end of the immobilised template. The primersmay be reversibly blocked to prevent extension. The single strandedtemplates may be sequenced using a hybridised primer at the 3′ end. Thesequencing primer may be removed after sequencing, and the immobilisedprimers deblocked to release an extendable 3′ hydroxyl. These primersmay be used to copy the template using bridged strand resynthesis toproduce a second immobilised template that is complementary to thefirst. Removal of the first template from the surface allows the newlysingle stranded second template to be sequenced, again from the 3′ end.Thus both ends of the original immobilised template can be sequenced.Such a technique allows paired end reads where the templates areamplified using a single extendable immobilised primer, for example asdescribed in Polony technology (Nucleic Acids Research 27, 24,e34(1999)) or emulsion PCR (Science 309, 5741, 1728-1732 (2005); Nature437, 376-380 (2005)). The details described herein below regarding theindividual steps apply mutatis mutandis to the steps described above,for example the primers may be blocked using the same techniques asdescribed in the relevant sections below.

If the amplification is performed on beads, either with a single ormultiple extendable primers, the beads may be analysed in solution, inindividual wells of a microtitre or picotitre plate, immobilised inindividual wells, for example in a fibre optic type device, orimmobilised as an array on a solid support. The solid support may be aplanar surface, for example a microscope slide, wherein the beads aredeposited randomly and held in place with a film of polymer, for exampleagarose or acrylamide.

When referring to immobilisation or attachment of molecules (e.g.nucleic acids) to a solid support, the terms “immobilised” and“attached” are used interchangeably herein and both terms are intendedto encompass direct or indirect, covalent or non-covalent attachment,unless indicated otherwise, either explicitly or by context. In certainembodiments of the invention covalent attachment may be preferred, butgenerally all that is required is that the molecules (e.g. nucleicacids) remain immobilised or attached to the support under theconditions in which it is intended to use the support, for example inapplications requiring nucleic acid amplification and/or sequencing.

Certain embodiments of the invention may make use of solid supportscomprised of an inert substrate or matrix (e.g. glass slides, polymerbeads etc) which is been “functionalised”, for example by application ofa layer or coating of an intermediate material comprising reactivegroups which permit covalent attachment to biomolecules, such aspolynucleotides. Examples of such supports include, but are not limitedto, polyacrylamide hydrogels supported on an inert substrate such asglass. In such embodiments, the biomolecules (e.g. polynucleotides) maybe directly covalently attached to the intermediate material (e.g. thehydrogel) but the intermediate material may itself be non-covalentlyattached to the substrate or matrix (e.g. the glass substrate). The term“covalent attachment to a solid support” is to be interpretedaccordingly as encompassing this type of arrangement.

In order to sequence two regions of a given target double-strandedpolynucleotide using the methods of the invention, the clusters can bemanipulated either to excise the region of the target DNA between thetwo ends, or to obtain sequence data from both strands of the duplex. Ifthe central portion of the cluster is excised, and the ends joined, itis possible to read the shortened contiguous sequence of the cluster ina single sequencing reaction that reads across both ends of the originaltemplate. This is similar to the idea of vector based (ditag) cloning insolution, but uses the cluster on the surface, which comprises a singlesequence to ensure the two ends of the original fragment are joined. Inthe case of vectors in solution, the DNA fragments are ligated intocircular constructs, and therefore the two ends (or tags) of thefragments remain on the same molecule even when the circle is cut open.This ensures two ends from the same original fragment are joined backtogether (to form ditags), without inter-molecular scrambling. In thecase of clusters, the fact that the ends of the duplex fragments remainattached to the surface ensures that the ends from the original fragmentare joined, which also prevents sequences from different molecules, orclusters, from mixing together. See FIG. 3 for a schematic of paired-endsequencing that calls for ligation of shortened ends. At the start ofthe procedure, the two ends A and B are covalently linked together via aseries of intermediate bases, for example 100-1000 bases. A firsttreatment step cuts the cluster strands to leave only the ends A and Bas separate short duplexes that are no longer covalently connected. Asingle strand of each duplex is attached to the surface, with the otherstrand hybridised. If the duplexes are made blunt ended, and ligatedback together, ends AB (and AA and BB) are rejoined covalently withoutthe intermediate bases.

Methods of treating the duplex to cut the strands at specific, butsequence independent, positions include using a restriction enzyme.Suitable restrictions enzymes are well known in the prior art, forexample Mme1, that cuts 18 and 20 bases remotely to its binding site. Itis desirable for the restriction enzyme to reach as far as possible intothe unknown region of the DNA, and an enzyme that cuts between 10-50bases, or further, is preferred. In a particular embodiment, an enzymethat cuts between 15-30 bases remote from its binding site, such asEcoP15, is envisioned.

The recognition sites for the particular restriction enzyme used can beengineered into both the primer used for solid phase amplification, andattached to the ends of the target fragments in the sample preparationstep. The attachment of universal known ends to a library of DNAfragments by ligation allows the amplification of a large variety ofdifferent sequences in a single amplification reaction. The sequences ofthe known sequence portion of the nucleic acid template can be designedsuch the type 2s restriction enzymes bind to the known region, and cutinto the unknown region of the amplified template.

Treatment of the duplexes where both strands are immobilised with therestriction enzyme(s) will give rise to two shortened duplex fragmentsin each cluster, each duplex anchored to the surface by one of its5′-ends. Treatment with restriction enzymes usually leaves an overhangon one of the strands. The single stranded overhang can be filled in orremoved, with a polymerase or exonuclease respectively, to ensure theends of the duplex are blunt.

The blunt ended duplex fragments can then be joined together by ligationto produce duplexes where both 5′ ends are immobilised. Such shortenedduplexes contain the sequences derived from the two ends of originalfragment, but without any intervening bases. A single sequencing run ofsay, 50 bases, could obtain the sequence of 25 bases from each end ofthe original fragment.

To facilitate sequencing, it is preferable if one of the strands isremoved from the surface to allow efficient hybridisation of asequencing primer to the remaining immobilised strand. Suitable methodsfor linearisation are described below, and described in more detail inapplication number WO07010251, the contents of which are incorporatedherein by reference in their entirety. Linearisation also serves tolimit the problem of ligation of two fragments from the same end ratherthan opposing ends. For each fragment with ends A and B, the ‘stumps’ Aand B are left on the surface after the cleavage step. There is nothingto prevent A ligating to A or B ligating to B to give two repeatedcopies of one end, rather than the desired one copy of two ends. Thelinearisation of one of the strands helps avoid this problem, as for thethree possibilities A-A, A-B or B-B, one duplex will have both strandsremoved, and one will have neither removed, and therefore remain as aduplex that is unavailable for hybridisation. Methods for linearisationand sequencing of clusters are described in full below.

In the first method of the invention, sequence data can be obtained fromboth ends of the immobilised duplex by a method wherein the duplex istreated to free a 3′-hydroxyl moiety that can be used an extensionprimer. The extension primer can then be used to read the first sequencefrom one strand of the template. After the first read, the strand can beextended to fully copy all the bases up to the end of the first strand.This second copy remains attached to the surface at the 5′-end. If thefirst strand is removed from the surface, the sequence of the secondstrand can be read. This gives a sequence read from both ends of theoriginal fragment.

In the third method of the invention, sequence data can be obtained fromboth ends of a template duplex by obtaining a sequence read from onestrand of the template from a primer in solution, copying the strandusing immobilised primers, releasing the first strand and sequencing thesecond, copied strand.

Methods of generating a free 3′-hydroxyl in only one strand of a dupleximmobilised at both ends include either treatment with a nicking enzyme,or chemical treatment to remove a specific nucleotide. Suitable nickingenzymes are well known in the art, and would preferably cut at a sitethat is 3′-remote to their binding site to avoid having to sequencebases that derive from the known nicked site. The nicking enzyme shouldcut only one of the immobilised strands, at the end closest to thesurface. See FIG. 1 for schematic of paired end sequencing methodologyutilising a nicking enzyme. Examples of suitable restriction enzymeswould include Nt.BstNBI and Nt.AlwI, which have no bases of definedsequence beyond the released 3′-hydroxyl.

Methods of removing nucleotides from the duplex involve the design ofthe template such that the base immediately adjacent to the unknowntarget region can be removed to make an abasic site. An “abasic site” isdefined as a nucleotide position in a polynucleotide chain from whichthe base component has been removed. Abasic sites can occur naturally inDNA under physiological conditions by hydrolysis of nucleotide residues,but may also be formed chemically under artificial conditions or by theaction of enzymes. Once formed, abasic sites may be cleaved (e.g. bytreatment with an endonuclease or other single-stranded cleaving enzyme,exposure to heat or alkali), providing a means for site-specificcleavage of a polynucleotide strand.

In a non-limiting embodiment an abasic site may be created at apre-determined position on one strand of a template polynucleotideduplex and then cleaved by first incorporating deoxyuridine (U) at apre-determined cleavage site in one strand of the templatepolynucleotide duplex. This can be achieved, for example, by including Uin one of the primers used for preparation of the templatepolynucleotide duplex by solid-phase amplification. The enzyme uracilDNA glycosylase (UDG) may then be used to remove the uracil base,generating an abasic site on one strand. The polynucleotide strandincluding the abasic site may then be cleaved at the abasic site bytreatment with endonuclease (e.g EndoIV endonuclease, AP lyase, FPGglycosylase/AP lyase, EndoVIII glycosylase/AP lyase), heat or alkali. Ina particular embodiment, the USER reagent available from New EngladBiolabs (M5505S) is used for the creation of a single nucleotide gap ata uracil base in a duplex strand. Treatment with endonuclease enzymesgives rise to a 3′-phosphate moiety at the cleavage site, which can beremoved with a suitable phosphatase such as alkaline phosphatase. SeeFIG. 2 for a schematic of a paired-end sequencing reaction that utilizesUSER linearization resynthesis.

Abasic sites may also be generated at non-natural/modifieddeoxyribonucleotides other than deoxyuridine and cleaved in an analogousmanner by treatment with endonuclease, heat or alkali. For example,8-oxo-guanine can be converted to an abasic site by exposure to FPGglycosylase. Deoxyinosine can be converted to an abasic site by exposureto AlkA glycosylase. The abasic sites thus generated may then becleaved, typically by treatment with a suitable endonuclease (e.g.EndoIV, AP lyase). A further example includes the use of a specific nonmethylated cytosine. If the remainder of the primer sequences, and thedNTP's used in cluster formation are methylated cytosine residues, thenthe non-methylated cytosines can be specifically converted to uracilresidues by treatment with bisulfite. This allows the use of the same‘USER’ treatment to linearise both strands of the cluster, as one of theprimers may contain a uracil, and one may contain the cytosine that canbe converted into a uracil (effectively a ‘protected uracil’ species).If the non-natural/modified nucleotide is to be incorporated into anamplification primer for use in solid-phase amplification, then thenon-natural/modified nucleotide should be capable of being copied by thepolymerase used for the amplification reaction.

In one embodiment, the molecules to be cleaved may be exposed to amixture containing the appropriate glycosylase and one or more suitableendonucleases. In such mixtures the glycosylase and the endonucleasewill typically be present in an activity ratio of at least about 2:1.

This method of cleavage has particular advantages in relation to thecreation of templates for nucleic acid sequencing. In particular,cleavage of an abasic site generated by treatment with a reagent such asUSER automatically releases a free 3′ phosphate group on the cleavedstrand which after phosphatase treatment can provide an initiation pointfor sequencing a region of the complementary strand. Moreover, if theinitial double-stranded nucleic acid contains only one cleavable (e.g.uracil) base on one strand then a single “nick” can be generated at aunique position in this strand of the duplex. Since the cleavagereaction requires a residue, e.g. deoxyuridine, which does not occurnaturally in DNA, but is otherwise independent of sequence context, ifonly one non-natural base is included there is no possibility ofglycosylase-mediated cleavage occurring elsewhere at unwanted positionsin the duplex. In contrast, were the double-stranded nucleic acid to becleaved with a “nicking” endonuclease that recognises a specificsequence, there is a possibility that the enzyme may create nicks at“other” sites in the duplex (in addition to the desired cleavage site)if these possess the correct recognition sequence. This could present aproblem if nicks are created in the strand it is intended to copy,rather than the strand that will be fully or partially removed, and is aparticular risk if the target portion of the double-stranded nucleicacid molecule is of unknown sequence. These limitations may be overcomeby preparing a sample with the recognition site for two differentnicking enzymes. If both preparations are processed separately using themethod, the regions of the genome cleaved by one nicking enzyme shouldbe represented in the other preparation.

The fact that there is no requirement for the non-natural (e.g. uracil)residue to be located in a detailed sequence context in order to providea site for cleavage using this approach is itself advantageous. Inparticular, if the cleavage site is to be incorporated into anamplification primer to be used in the production of a clustered arrayby solid-phase amplification, it is only necessary to replace onenatural nucleotide (e.g. T) in the primer with a non-natural nucleotide(e.g. U) in order to enable cleavage. There is no need to engineer theprimer to include a restriction enzyme recognition sequence of severalnucleotides in length. Oligonucleotide primers including U nucleotides,and other non-natural nucleotides, such as those listed above, caneasily be prepared using conventional techniques and apparatus forchemical synthesis of oligonucleotides.

Another advantage gained by cleavage of abasic sites in adouble-stranded molecule generated by action of UDG on uracil is thatthe first base incorporated in a “sequencing-by-synthesis” reactioninitiating at the free 3′ hydroxyl group formed by cleavage at such asite will always be T. Hence, if the template polynucleotide duplexforms part of a clustered array comprised of many such molecules, all ofwhich are cleaved in this manner to produce sequencing templates, thenthe first base universally incorporated across the whole array will beT. This can provide a sequence-independent assay for individual clusterintensity at the start of a sequencing “run”.

In particular cases, it may be advantageous to perform a blockingtreatment with a dideoxynucleotide triphosphate and a polymerase and/orterminal transferase. After a solid phase amplification, there remain onthe surface a large number of unused amplification primers, in additionto the free 3′-ends of each of the template strands. Treatment with sucha blocked nucleotide ensures these free 3′-OH functional groups areunavailable for extension during any subsequent polymerase steps. Inmethods where 3′-hydroxyl groups are created with chemical treatment orrestriction endonucleases, it is advantageous to block any residual3′-hydroxyl groups before the desired 3′-hydroxyl groups are created. Inthe case of treatment with the USER reagent, as a phosphate group isreleased, the blocking step can be performed either before or after theUSER treatment, as the phosphate group will act as a protecting group toprevent blocking of the desired 3′-hydroxyl moieties. The phosphategroup can be removed after the blocking step has been performed.

The act of restriction enzyme treatment or abasic site generation andcleavage results in a free 5′-end on the strand that is no longerimmobilised to the surface. This strand can be completely removed fromthe surface by treatment with a 5′-3′ exonuclease, such as lambda or T7exonuclease. Such a treatment means that the template strand is singlestranded, and available for subsequent copying. If the polymerase thatis used to extend the 3′-hydroxyl group has a strand displacingactivity, then the 5′-3′ exonuclease treatment may not be necessary.

In the embodiment of the invention, using an immobilised primer forsequencing, it is advantageous to extend the free 3′-hydroxyl primerwith a plurality of bases complementary to the template prior toinitiating sequencing. This both raises the melting temperature of theimmobilised duplex, and helps prevent the template strand fromre-hybridising to other immobilised primers during sequencing, whichgives rises to phasing problems within the cluster. The ligation step iscarried out after the phosphatase step has removed the phosphate groupfrom the immobilised primer. Addition of 20-30 bases of sequence can beperformed by a ligation reaction with a 5′-phosphate modified primerhydridised adjacent to the free 3′-hydroxyl. A ligase, such as T4 DNAligase can be used to seal the gap. In the case of USER treatment whichremoves the U nucleotide, the 5′-base of the primer will be T thatreplaces the excised U. For the hybridisation step to be carried outefficiently, the 5′-non immobilised strand must have been removed by5′-3′ exonucleolysis treatment as described above. Such immobilised,extended primers with a free 3′-hydroxyl are described as extended5′-anchored, or extended immobilised primers, and generation of suchextended primers is only one of the steps involved in treating theplurality of double stranded template polynucleotides such that thefirst template strand is hybridised to a primer that is immobilised onthe solid support at its 5′-end.

The free 3′-hydroxyl group on the 5′-anchored primer or extended primercan be used to initiate rounds of sequencing to determine the sequenceof the bases in the hybridised template. Sequencing can be carried outusing any suitable “sequencing-by-synthesis” technique, whereinnucleotides are added successively to the free 3′ hydroxyl group,resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. The nature of the nucleotide added is preferably determinedafter each addition. Alternative methods of sequencing includesequencing by ligation, for example as described in U.S. Pat. No.6,306,597 or WO06084132, the contents of which are incorporated hereinby reference.

The use of USER according to the present invention produces a linearisedfirst template strand and a lawn of primers left with phosphate blockingmoieties. Without further phosphatase treatment, the immobilised primerlawn is not suitable for polymerase extension. A sequencing primer insolution can be used to initiate a sequencing read for the firsttemplate strand. If the phosphate groups are removed from the lawn ofprimers, then the first template strands, once the first extendedsequencing primer has been removed, can be hybridised to the primer lawnand subjected to one or more cycles of extension, denaturation andre-hybridisation. The denaturation can be performed thermally orisothermally, for example using chemical denaturation. The chemicaldenaturatant may be urea, hydroxide or formamide or other similarreagent. This re-generates the template duplexes where both strands areimmobilised. The first strand can be removed by a suitable orthogonallinearisation treatment step, such as diol cleavage or removal of an8-oxo-G residue, and after denaturation of the first template strand, asecond sequencing primer can be hybridised to the second templatestrand, and the second template strand sequenced. This orthogonallinearisation strategy allows reads from both ends of the template.

The first method of the invention provides a sequencing reaction carriedout using an immobilised primer. The immobilised sequencing primer canbe denatured, and the first template strand used to re-initiate furtherrounds of extension or amplification as above. Again this re-generatesthe template duplexes where both strands are immobilised. The firststrand can be removed by a suitable orthogonal linearisation treatmentstep, such as diol cleavage or removal of an 8-oxo-G residue, and afterdenaturation of the first template strand, a second sequencing primercan be hybridised to the second template strand, and the second templatestrand sequenced. This orthogonal linearisation strategy also allowsreads from both ends of the template.

Both of the methods detailed above allow for cluster repopulationbetween the first and second reads. This is beneficial to increase thelevel of signal obtained for the second read of the cluster. Any methodthat results in both amplification primers being retained on the surfaceduring the first sequencing read is within the scope of the third methodof the invention, although it is advantageous if the primers can betreated, as in the case of USER, to make the 3′-hydroxyl unavailable forprimer extension during the sequencing reaction. An alternative methodof blocking the unextended grafting primers would be to use a nucleosidecarry an unextendable 3′ group. Treating the surface with such anucleotide and terminal transferase would block the surface to furtherextension. After the first sequencing run, the nucleotides could bedeprotected to allow further cycles of copying the template strand. Ifthe nucleotides carry a dideoxy modification, this can be removed usingan exonuclease, or a polymerase with exonuclease activity. Thus theclusters could be made with two grafted primers as described, the unusedprimers blocked during the first sequencing reaction, then deblocked toallow further copying of the template strands.

A portion of the amplification primers may be attached to the surfacewith a modification blocking the 3′hydroxyl from extension in theamplification cycles. This in effect means that the surface is treatedwith three or more amplification primers rather than two. At least twoof the amplification primers should comprise regions of identicalsequence, but at least one primer will not be susceptible to theconditions used to remove the second primer during the linearisationprocess, and will contain a 3′-blocking moiety. The blocking moiety maytake the form of a chemical block, such as an azidomethyl group that canbe removed with a phosphine reagent, an enzymatically removable such asa phosphate group that can be removed with a phosphatase, or may be inthe form of a nucleoside group that can be removed using 3′-5′exonucleolysis. Such nucleoside modifications include abasic sites, thatcan be removed as described, or 2′, 3′ dideoxy nucleotides that can beremoved by a polymerase with exonuclease activity. Further modificationsinclude using an oligonucleotide sequence that can form a selfcomplementary region with a recognition sequence for a restrictionenzyme, as shown in FIG. 11. Treatment with the restriction enzymeshould cut the hairpin strand and release a shorter sequence with a free3′ hydroxyl group. Treatment of the surface after the first sequencingrun is completed to deblock the primers will allow the remaining firststrand to hybridise to the deprotected primers and recopy the alreadysequenced strands. A second sequencing read can be obtained by removingthe first strand, hybridising a sequencing primer and reading the newlysynthesised second strand.

One particular sequencing method which can be used in the methods of theinvention relies on the use of modified nucleotides that can act asreversible chain terminators. Such reversible chain terminators compriseremovable 3′ blocking groups. Once such a modified nucleotide has beenincorporated into the growing polynucleotide chain complementary to theregion of the template being sequenced there is no free 3′-OH groupavailable to direct further sequence extension and therefore thepolymerase can not add further nucleotides. Once the nature of the baseincorporated into the growing chain has been determined, the 3′ blockmay be removed to allow addition of the next successive nucleotide. Byordering the products derived using these modified nucleotides it ispossible to deduce the DNA sequence of the DNA template. Such reactionscan be done in a single experiment if each of the modified nucleotideshas attached thereto a different label, known to correspond to theparticular base, to facilitate discrimination between the bases added ateach incorporation step. Suitable labels are described in copending PCTapplication PCT/GB/2007/001770, the contents of which are incorporatedherein by reference in their entirety. Alternatively, a separatereaction may be carried out containing each of the modified nucleotidesadded individually.

The modified nucleotides may carry a label to facilitate theirdetection. In a particular embodiment, the label is a fluorescent label.Each nucleotide type may carry a different fluorescent label. Howeverthe detectable label need not be a fluorescent label. Any label can beused which allows the detection of the incorporation of the nucleotideinto the DNA sequence.

One method for detecting the fluorescently labelled nucleotidescomprises using laser light of a wavelength specific for the labellednucleotides, or the use of other suitable sources of illumination. Thefluorescence from the label on an incorporated nucleotide may bedetected by a CCD camera or other suitable detection means. Suitabledetection means are described in copending applicationPCT/US2007/007991, the contents of which are incorporated herein byreference in their entirety.

Once the first sequencing read is complete, and sufficient read lengthhas been determined, the rest of the strand can be copied. If the3′-hydroxyl group was originally created with a nicking enzyme, then itwill be possible to re-create a fresh 3′-hydroxyl group at the sameposition, and extend from this position, however it is equally possibleto continue to copy the first template strand from the 3′-hydroxyl groupof the nucleotides incorporated as part of the sequencing reaction. Thisextension reaction with all four unlabelled nucleotides and a polymerasewill copy all the bases of first template. The immobilised primers maycontain a restriction site for a nicking enzyme, and treatment with therestriction enzyme may shorten the immobilised primers, or theimmobilised template duplexes to release an unblocked 3′ hydroxyl group,as shown in FIG. 9.

In the course of developing the protocols included herein, it wassurprisingly noticed that sequencing results were improved in situationswhere the 5′ end of the template was capable of hybridisation to animmobilised primer. In short, sequencing as shown in FIG. 4 c was betterthan sequencing as shown in FIG. 4 a. In the experimental results shownherein, better means that the level of signal retained over multiplecycles was higher, meaning that the decay curve with both endsimmobilised was less steep, and the signal higher than that where only asingle end is immobilised. This may be due to the fact that any templatestrand breakages that occur during the sequencing process do notautomatically result in loss of the template from the surface, as the 3′end of the template is also held in place by hybridisation, althoughthere may also be other factors that cause this effect.

The first template strand may be attached to the surface in a way thatallows selective removal. If the first template strand is removed fromthe surface, and the duplex strands denatured, for example by treatmentwith hydroxide or formamide, then the second, copied strand remainsimmobilised as a linearised single strand. As the end sequences of thisstrand are known, it is possible to hybridise a sequencing primer to thesecond template strand, and by repeating the cycles of sequencing asdescribed above, determine the sequence of a second read for the otherend of the template to the first read.

Selective removal, or linearisation of the first template strand can beachieved in a number of ways. The linearization to allow hybridizationof a sequencing primer in solution does not have to leave a functional3′-hydroxyl on the template strand, and can cleave either one strand orboth strands. Thus, as used herein, the term “linearization” refers tothe selective removal of a complementary strand. If one of theamplification primers is immobilised such that it can be cleaved fromthe surface, the resulting double stranded DNA can be made singlestranded using heat or chemical denaturing conditions to give a singlestranded molecule containing a primer hybridisation site. The singlestranded molecule can be hybridised with a sequencing primer in solutionto allow a sequencing read of the immobilised template strand. Saidcleavage site is a site which allows controlled cleavage of the firsttemplate strand by chemical, enzymatic or photochemical means.

Any suitable enzymatic, chemical or photochemical cleavage reaction maybe used to cleave. A number of suitable methods are described inWO07010251, the contents of which are incorporated herein by referencein their entirety. The cleavage reaction may result in removal of a partor the whole of the strand being cleaved. Suitable cleavage meansinclude, for example, restriction enzyme digestion, in which case thecleavage site is an appropriate restriction site for the enzyme whichdirects cleavage of one or both strands of a duplex template; RNasedigestion or chemical cleavage of a bond between a deoxyribonucleotideand a ribonucleotide, in which case the cleavage site may include one ormore ribonucleotides; chemical reduction of a disulphide linkage with areducing agent (e.g. TCEP), in which case the cleavage site shouldinclude an appropriate disulphide linkage; chemical cleavage of a diollinkage with periodate, in which case the cleavage site should include adiol linkage; generation of an abasic site and subsequent hydrolysis,etc.

In one embodiment cleavage may occur at a cleavage site in one or bothstrands of a template polynucleotide duplex which comprises one or moreor any combination of non-natural nucleotides, ribonucleotides or anon-nucleotide chemical modifications.

Suitable cleavage techniques for use in the method of the inventioninclude, but are not limited to, the following:

i) Chemical Cleavage

The term “chemical cleavage” encompasses any method which utilises anon-nucleic acid and non-enzymatic chemical reagent in order topromote/achieve cleavage of one or both strands of a templatepolynucleotide duplex. If required, one or both strands of the templatepolynucleotide duplex may include one or more non-nucleotide chemicalmoieties and/or non-natural nucleotides and/or non-natural backbonelinkages in order to permit a chemical cleavage reaction. In aparticular embodiment, the modification(s) required to permit chemicalcleavage may be incorporated into an amplification primer used to formthe template polynucleotide duplex by solid-phase nucleic acidamplification.

In a preferred but non-limiting embodiment, one strand of the templatepolynucleotide duplex (or the amplification primer from which thisstrand is derived if formed by solid-phase amplification) may include adiol linkage which permits cleavage by treatment with periodate (e.g.,sodium periodate). It will be appreciated that more than one diol can beincluded at the cleavage site.

Diol linker units based on phosphoamidite chemistry suitable forincorporation into polynucleotide chains are commercially available fromFidelity systems Inc. (Gaithersburg, Md., USA). One or more diol unitsmay be incorporated into a polynucleotide using standard methods forautomated chemical DNA synthesis. Hence, oligonucleotide primersincluding one or more diol linkers can be conveniently prepared bychemical synthesis.

In order to position the diol linker at an optimum distance from thesolid support, one or more spacer molecules may be included between thediol linker and the site of attachment to the solid support. The spacermolecule may be a non-nucleotide chemical moiety. Suitable spacer unitsbased on phosphoamidite chemistry for use in conjunction with diollinkers are also supplied by Fidelity Systems, Inc. One suitable spacerfor use with diol linkers is the spacer denoted arm 26, identified inthe accompanying examples. To enable attachment to a solid support atthe 5′ end of the polynucleotide strand, arm 26 may be modified toinclude a phosphorothioate group. The phosphorothioate group can easilybe attached during chemical synthesis of a “polynucleotide” chainincluding the spacer and diol units.

Other spacer molecules could be used as an alternative to arm 26. Forexample, a stretch of non-target “spacer” nucleotides may be included.Typically from 1 to 20, more particularly from 1 to 15 or from 1 to 10,and even more particularly 2, 3, 4, 5, 6, 7, 8, 9 or 10 spacernucleotides may be included. In a particular embodiment, 10 spacernucleotides are positioned between the point of attachment to the solidsupport and the diol linker. In another particular embodiment, polyTspacers are used, although other nucleotides and combinations thereofcan be used. In another particular embodiment, the primer may include10T spacer nucleotides.

The diol linker is cleaved by treatment with a “cleaving agent”, whichcan be any substance which promotes cleavage of the diol. In aparticular embodiment, the cleaving agent is periodate, preferablyaqueous sodium periodate (NaIO₄). Following treatment with the cleavingagent (e.g., periodate) to cleave the diol, the cleaved product may betreated with a “capping agent” in order to neutralise reactive speciesgenerated in the cleavage reaction. Suitable capping agents for thispurpose include amines, such as ethanolamine or propanolamine.Advantageously, the capping agent (e.g., propanolamine) may be includedin a mixture with the cleaving agent (e.g., periodate) so that reactivespecies are capped as soon as they are formed.

The combination of a diol linkage and cleaving agent (e.g., periodate)to achieve cleavage of at least one strand of a template polynucleotideduplex may be used to particular advantage for linearisation of templateduplexes on solid supported polyacrylamide hydrogels because treatmentwith periodate is compatible with nucleic acid integrity and with thechemistry of the hydrogel surface. Utility of diol linkages/periodate asa method of linearisation is not, however, limited to polyacrylamidehydrogel surfaces but also extends to linearisation of duplexesimmobilised on other solid supports and surfaces, including supportscoated with functionalised silanes (etc).

In a further embodiment, the strand to be cleaved (or the amplificationprimer from which this strand is derived if prepared by solid-phaseamplification) may include a disulphide group which permits cleavagewith a chemical reducing agent, e.g. Tris (2-carboxyethyl)-phosphatehydrochloride (TCEP).

ii) Cleavage of Abasic Sites

The use of abasic sites is described above in order to generate a free3′-hydroxyl moiety to act as a sequencing primer. If both amplificationprimers are modified such that they can be sequentially cleaved, thesecond cleavage can be used to cleave the first strand from the surface.The first (or second) primer could contain a uracil base, that can becleaved by one enzyme (UDG), and the second (or first) primer couldcontain an 8-oxo-guanine base that can be cleaved by a second,orthogonal enzyme, FPG glycosylase. The second abasic site cleavagecould be used to leave a sequencing primer attached to a surface, suchthat a G base is incorporated as the first cycle of sequencing, or thecleaved duplex strands can be denatured to allow hybridisation of asequencing primer in solution.

iii) Cleavage of Ribonucleotides

Incorporation of one or more ribonucleotides into a polynucleotidestrand which is otherwise comprised of deoxyribonucleotides (with orwithout additional non-nucleotide chemical moieties, non-natural basesor non-natural backbone linkages) can provide a site for cleavage usinga chemical agent capable of selectively cleaving the phosphodiester bondbetween a deoxyribonucleotide and a ribonucleotide or using aribonuclease (RNAse). Therefore, sequencing templates can be produced bycleavage of one strand of a template polynucleotide duplex at a sitecontaining one or more consecutive ribonucleotides using such a chemicalcleavage agent or an RNase. In a particular embodiment, the strand to becleaved contains a single ribonucleotide, which provides a site forchemical cleavage.

Suitable chemical cleavage agents capable of selectively cleaving thephosphodiester bond between a deoxyribonucleotide and a ribonucleotideinclude metal ions, for example rare-earth metal ions (especially La³⁺,particularly Tm³⁺, Yb³⁺or Lu³⁺ (Chen et al. Biotechniques. 2002, 32:518-520; Komiyama et al. Chem. Commun. 1999, 1443-1451)), Fe(3) orCu(3), or exposure to elevated pH, e.g., treatment with a base such assodium hydroxide. By “selective cleavage of the phosphodiester bondbetween a deoxyribonucleotide and a ribonucleotide” is meant that thechemical cleavage agent is not capable of cleaving the phosphodiesterbond between two deoxyribonucleotides under the same conditions.

The base composition of the ribonucleotide(s) is generally not material,but can be selected in order to optimise chemical (or enzymatic)cleavage. By way of example, rUMP or rCMP are generally preferred ifcleavage is to be carried out by exposure to metal ions, especially rareearth metal ions.

The ribonucleotide(s) will typically be incorporated into one strand ofa template polynucleotide duplex (or the amplification primer from whichthis strand is derived if prepared by solid-phase amplification), andmay be situated in a region of the duplex which is single-stranded whenthe two complementary strands of the duplex are annealed (i.e., in a 5′overhanging portion). If the template polynucleotide duplex is preparedby solid-phase amplification using forward and reverse amplificationprimers, one of which contains at least one ribonucleotide, the standardDNA polymerase enzymes used for amplification are not capable of copyingribonucleotide templates. Hence, the amplification products will containan overhanging 5′ region comprising the ribonucleotide(s) and anyremainder of the amplification primer upstream of the ribonucleotide(s).

The phosphodiester bond between a ribonucleotide and adeoxyribonucleotide, or between two ribonucleotides may also be cleavedby an RNase. Any endolytic ribonuclease of appropriate substratespecificity can be used for this purpose. If the ribonucleotide(s) arepresent in a region which is single-stranded when the two complementarystrands of the double-stranded molecule are annealed (i.e., in a 5′overhanging portion), then the RNase will be an endonuclease which hasspecificity for single strands containing ribonucleotides. For cleavagewith ribonuclease it is preferred to include two or more consecutiveribonucleotides, and preferably from 2 to 10 or from 5 to 10 consecutiveribonucleotides. The precise sequence of the ribonucleotides isgenerally not material, except that certain RNases have specificity forcleavage after certain residues. Suitable RNases include, for example,RNaseA, which cleaves after C and U residues. Hence, when cleaving withRNaseA the cleavage site must include at least one ribonucleotide whichis C or U.

Polynucleotides incorporating one or more ribonucleotides can be readilysynthesised using standard techniques for oligonucleotide chemicalsynthesis with appropriate ribonucleotide precursors. If the templatepolynucleotide duplex is prepared by solid-phase nucleic acidamplification, then it is convenient to incorporate one or moreribonucleotides into one of the primers to be used for the amplificationreaction.

iv) Photochemical Cleavage

The term “photochemical cleavage” encompasses any method which utiliseslight energy in order to achieve cleavage of one or both strands of thedouble-stranded nucleic acid molecule.

A site for photochemical cleavage can be provided by a non-nucleotidechemical spacer unit in one of the strands of the double-strandedmolecule (or the amplification primer from which this strand is derivedif prepared by solid-phase amplification). Suitable photochemicalcleavable spacers include the PC spacer phosphoamidite(4-(4,4′-Dimethoxytrityloxy)butyramidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite)supplied by Glen Research, Sterling, Va., USA (cat number 10-4913-XX)which has the structure:

The spacer unit can be cleaved by exposure to a UV light source.

This spacer unit can be attached to the 5′ end of a polynucleotide,together with a thiophosphate group which permits attachment to a solidsurface, using standard techniques for chemical synthesis ofoligonucleotides. Conveniently, this spacer unit can be incorporatedinto a forward or reverse amplification primer to be used for synthesisof a photocleavable template polynucleotide duplex by solid-phaseamplification.

v) PCR Stoppers

In another embodiment of the invention the template polynucleotideduplex may be prepared by solid-phase amplification using forward andreverse primers, one of which contains a “PCR stopper”. A “PCR stopper”is any moiety (nucleotide or non-nucleotide) which prevents read-throughof the polymerase used for amplification, such that it cannotextend/copy beyond that point. The result is that amplified strandsderived by extension of the primer containing the PCR stopper willcontain a 5′ overhanging portion. This 5′ overhang (other than the PCRstopper itself) may be comprised of naturally occurringdeoxyribonucleotides, with predominantly natural backbone linkages,i.e., it may simply be a stretch of single-stranded DNA. The moleculemay then be cleaved in the 5′ overhanging region with the use of acleavage reagent (e.g., an enzyme) which is selective for cleavage ofsingle-stranded DNA but not double stranded DNA, for example mung beannuclease.

The PCR stopper may be essentially any moiety which preventsread-through of the polymerase to be used for the amplificationreaction. Suitable PCR stoppers include, but are not limited to,hexaethylene glycol (HEG), abasic sites, and any non-natural or modifiednucleotide which prevents read-through of the polymerase, including DNAanalogues such as peptide nucleic acid (PNA).

Stable abasic sites can be introduced during chemical oligonucleotidesynthesis using appropriate spacer units containing the stable abasicsite. By way of example, abasic furan(5′-O-Dimethoxytrityl-1′,2′-Dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)spacers commercially available from Glen Research, Sterling, Va., USA,can be incorporated during chemical oligonucleotide synthesis in orderto introduce an abasic site. Such a site can thus readily be introducedinto an oligonucleotide primer to be used in solid-phase amplification.If an abasic site is incorporated into either forward or reverseamplification primer the resulting amplification product will have a 5′overhang on one strand which will include the abasic site (insingle-stranded form). The single-stranded abasic site may then becleaved by the action of a suitable chemical agent (e.g. exposure toalkali) or an enzyme (e.g. AP-endonuclease VI, Shida et al. NucleicAcids Research, 1996, Vol. 24, 4572-4576).

vi) Cleavage of Peptide Linker

A cleavage site can also be introduced into one strand of a templatepolynucleotide duplex by preparing a conjugate structure in which apeptide molecule is linked to one strand of the duplex (or theamplification primer from which this strand is derived if prepared bysolid-phase amplification). The peptide molecule can subsequently becleaved by a peptidase enzyme of the appropriate specificity, or anyother suitable means of non-enzymatic chemical or photochemicalcleavage. Typically, the conjugate between peptide and nucleic acid willbe formed by covalently linking a peptide to one strand only of thetemplate polynucleotide duplex, with the peptide portion beingconjugated to the 5′ end of this strand, adjacent to the point ofattachment to the solid surface. If the template polynucleotide duplexis prepared by solid-phase amplification, the peptide conjugate may beincorporated at the 5′ end of one of the amplification primers.Obviously the peptide component of this primer will not be copied duringamplification, hence the “bridged” amplification product will include acleavable 5′ peptide “overhang” on one strand.

Conjugates between peptides and nucleic acids wherein the peptide isconjugated to the 5′ end of the nucleic acid can be prepared usingtechniques generally known in the art. In one such technique the peptideand nucleic acid components of the desired amino acid and nucleotidesequence can be synthesised separately, e.g. by standard automatedchemical synthesis techniques, and then conjugated in aqueous/organicsolution. By way of example, the OPeC™ system commercially availablefrom Glen Research is based on the “native ligation” of an N-terminalthioester-functionalized peptide to a 5′-cysteinyl oligonucleotide.Pentafluorophenyl S-benzylthiosuccinate is used in the final couplingstep in standard Fmoc-based solid-phase peptide assembly. Deprotectionwith trifluoroacetic acid generates, in solution, peptides substitutedwith an N-terminal S-benzylthiosuccinyl group.O-trans-4-(N-a-Fmoc-S-tert-butylsulfenyl-1-cysteinyl)aminocyclohexylO-2-cyanoethyl-N,N-diisopropylphosphoramidite is used in the finalcoupling step in standard phosphoramidite solid-phase oligonucleotideassembly. Deprotection with aqueous ammonia solution generates insolution 5′-S-tert-butylsulfenyl-L-cysteinyl functionalizedoligonucleotides. The thiobenzyl terminus of the modified peptide isconverted to the thiophenyl analogue by the use of thiophenol, whilstthe modified oligonucleotide is reduced usingtris(carboxyethyl)-phosphine. Coupling of these two intermediates,followed by the “native ligation” step, leads to formation of theoligonucleotide-peptide conjugate.

The conjugate strand containing peptide and nucleic acid can becovalently attached to a solid support using any suitable covalentlinkage technique known in the art which is compatible with the chosensurface. If the peptide/nucleic acid conjugate structure is anamplification primer to be used for solid-phase amplification,attachment to the solid support must leave the 3′ end of the nucleicacid component free.

The peptide component can be designed to be cleavable by any chosenpeptidase enzyme, of which many are known in the art. The nature of thepeptidase is not particularly limited, it is necessary only for thepeptidase to cleave somewhere in the peptide component. Similarly, thelength and amino acid sequence of the peptide component is notparticularly limited except by the need to be “cleavable” by the chosenpeptidase.

The length and precise sequence of the nucleic acid component is alsonot particularly limited, it may be of any desired sequence. If thenucleic acid component is to function as a primer in solid-phaseamplification, then its length and nucleotide sequence will be selectedto enable annealing to the template to be amplified.

vii) Enzymatic Digestion with Restriction Endonuclease/NickingEndonuclease

Cleavage of double-stranded polynucleotides with restrictionendonuclease is a technique in routine use in the art of molecularbiology. Nicking endonucleases are enzymes that selectively cleave or“nick” one strand of a polynucleotide duplex and are also well known inthe art of molecular biology. The invention is not limited with respectto the nature of the enzyme. Essentially any restriction or nickingendonuclease may be used, provided that a suitable recognition sequencecan be included at the cleavage site. In the case of the invention usingtwo sequencing reads, the choice of nicking enzyme will need to bedifferent to that used in the first cycle of extension, and this can beenabled using amplification primers with recognition sites for twodifferent enzymes.

The method of the invention is described in further detail as follows.

Any suitable solid support and any suitable attachment means known inthe art may be used, of which several are described by way of examplebelow. In a particular embodiment, linkage to the solid support isachieved via covalent attachment.

The polynucleotide duplexes will typically be formed from twocomplementary polynucleotide strands comprised of deoxyribonucleotidesjoined by phosphodiester bonds, but may additionally include one or moreribonucleotides and/or non-nucleotide chemical moieties and/ornon-naturally occurring nucleotides and/or non-naturally occurringbackbone linkages. In particular, the double-stranded nucleic acid mayinclude non-nucleotide chemical moieties, e.g. linkers or spacers, atthe 5′ end of one or both strands. By way of non-limiting example, thedouble-stranded nucleic acid may include methylated nucleotides, uracilbases, phosphorothioate groups, ribonucleotides, diol linkages,disulphide linkages, peptides etc. Such non-DNA or non-naturalmodifications may be included in order to permit cleavage, or to confersome other desirable property, for example to enable covalent attachmentto a solid support, or to act as spacers to position a site of cleavagean optimal distance from the solid support.

The template duplexes may also include non-target sequences at both the5′ and 3′ends, flanking the target polynucleotide. If the templateduplexes are formed by solid-phase amplification, these non-targetsequences will generally be derived from the primers used forsolid-phase amplification.

The polynucleotide duplexes form part of a single cluster or colonycomprised of many such first and second duplexes, and the cluster orcolony will itself typically form part of an array of many such clustersor colonies. The terms “cluster” and “colony” are used interchangeablythroughout and refer to a discrete site on a solid support comprised ofa plurality of identical immobilised nucleic acid strands and aplurality of identical immobilised complementary nucleic acid strands.The term “clustered array” refers to an array formed from such clustersor colonies.

A key feature of the invention is that both sequencing runs can occur inthe same cluster or colony on a clustered array. On such an array eachduplex within each colony will comprise the same double-stranded targetpolynucleotide, whereas different colonies may be formed of duplexescomprising different double-stranded target polynucleotides. In aparticular embodiment at least 90%, more particularly at least 95% ofthe colonies on a given clustered array will be formed from templateduplexes comprising different double-stranded target polynucleotides,although within each individual colony on the array all templateduplexes will comprise the same double-stranded target polynucleotide.

The amplified polynucleotides can be treated in such a way to enableextension of a hybridised primer. Each polynucleotide duplex on thearray contains the same universal primer recognition regions to allowthe same primers to be used to sequence every cluster. A firstsequencing primer is then hybridised to the first template strand and asequencing reaction proceeds via successive incorporation of nucleotidesor oligonucleotides to the first sequencing primer, resulting indetermination of the sequence of a first region of the targetpolynucleotide. In the case of the method where both ends of theoriginal fragment are made contiguous (see FIG. 3), the first sequencingrun gives sequence information for both ends of the original fragment.At this point, the method is complete; there is no need for furtherprocessing of the sample.

By contrast, the other methods of the invention require two sequencingreactions to be performed. The first sequencing reaction is initiatedeither by the 3′-hydroxyl group of the immobilised primer that is freedfrom within the immobilised duplex, or is initiated by a sequencingprimer added from solution. The second sequencing reaction is initiatedby a sequencing primer that can either be immobilised or applied insolution. Hybridisation of the sequencing primer in solution to thetemplate strand is achieved by contacting the primer and template strandunder conditions which promote annealing of primer to template. Suchconditions will generally be well known to those skilled in the art ofmolecular biology.

When the first sequencing reaction is complete, if the first sequencingprimer is immobilised this can be used to further extend the strand tocopy every base on the template. This is carried out using the fournative nucleotide triphosphates, dATP, dGTP, dCTP and dTTP, and asuitable polymerase. If the complementary strand was removed bytreatment with a 5′-3′ exonuclease step prior to the sequencingreaction, then the extension reaction can be carried out at anytemperature as the strand is already single stranded, and therefore anypolymerase such as Klenow, Taq or vent is suitable. If the strand hasnot been removed, then a polymerase with strand displacing activity,such as Bst polymerase will be required.

Prior to undertaking the extension reaction, it may be advantageous toextend the immobilised primer, as shown in FIG. 10. The extension canperformed using a hybridised oligonucleotide with a sequence thatextends beyond the 3′ end of the immobilised primer, whose sequence isalso the same sequence as the corresponding region at the end of thetemplate. This extended portion can serve as a basis for extension ofthe immobilised primer, and thus the extended primer is complementary tothe immobilised template strand. The extended primers may improve theefficiency of the strand resynthesis step due to their increased length.

Once a complementary sequence of the first strand has been generated,the first strand can be removed from the surface as described above. Asecond sequencing primer is then hybridised to the copied strand of thetemplate and a sequencing reaction proceeds via successive addition ofnucleotides to the second sequencing primer, resulting in determinationof the sequence of a second region of the target polynucleotide. SeeFIG. 1.

As described above, sequencing can be carried out using any suitable“sequencing-by-synthesis” technique, wherein nucleotides oroligonucleotides are added successively to a free 3′ hydroxyl group,typically provided by annealing of a sequencing primer, resulting insynthesis of a polynucleotide chain in the 5′ to 3′ direction. In aparticular embodiment, the nature of the nucleotide or oligonucleotideadded is determined after each addition.

One particular sequencing method which can be used in the methods of theinvention relies on the use of modified nucleotides that can act asreversible chain terminators. Nucleotides for use in the invention aredescribed fully in WO04018497. Once the modified nucleotide has beenincorporated into the growing polynucleotide chain complementary to theregion of the template being sequenced there is no free 3′-OH groupavailable to direct further sequence extension and therefore thepolymerase can not add further nucleotides. Once the nature of the baseincorporated into the growing chain has been determined, the 3′ blockmay be removed to allow addition of the next successive nucleotide. Byordering the products derived using these modified nucleotides it ispossible to deduce the DNA sequence of the DNA template. Such reactionscan be done in a single experiment if each of the modified nucleotideshas attached thereto a different label, known to correspond to theparticular base, which facilitates discrimination between the basesadded at each incorporation step. Alternatively, a separate reaction maybe carried out containing each of the modified nucleotides, which areadded separately.

The modified nucleotides may carry a label to facilitate theirdetection. In a particular embodiment, the label is a fluorescent label.Each nucleotide type may carry a different fluorescent label.Fluorescent labels suitable for use in the current invention aredescribed in PCT application PCT/GB2007/001770. However the detectablelabel need not be a fluorescent label. Any label can be used whichallows the detection of the incorporation of the nucleotide into the DNAsequence.

One method for detecting the fluorescently labelled nucleotidescomprises using laser light of a wavelength specific for the labellednucleotides, or the use of other suitable sources of illumination. Thefluorescence from the label on the nucleotide may be detected by a CCDcamera or other suitable detection means. An imaging system suitable fordetermining the fluorescence signal from incorporated nucleotides isdescribed in PCT application number PCT/US07/007991.

The methods of the invention are not limited to use of the sequencingmethod outlined above, but can be used in conjunction with essentiallyany sequencing methodology which relies on successive incorporation ofnucleotides into a polynucleotide chain. Suitable techniques include,for example, Pyrosequencing™, FISSEQ (fluorescent in situ sequencing),MPSS (massively parallel signature sequencing) and sequencing byligation-based methods.

The target double-stranded polynucleotide to be sequenced using themethod of the invention may be any polynucleotide that it is desired tosequence. The target polynucleotide may be of known, unknown orpartially known sequence,such as, for example in re-sequencingapplications. Using the template preparation method described in detailbelow it is possible to prepare arrays of templates starting fromessentially any double-stranded target polynucleotide of known, unknownor partially known sequence. With the use of arrays it is possible tosequence multiple targets of the same or different sequence in parallel.A particular application of the pairwise method is in the sequencing offragments of genomic DNA. The method provides particular advantages inthe identification of genome rearrangements, since the two regions ofsequence obtained for each target molecule using the method will beknown to be linked within a certain distance of each other in thegenome, depending on the size of the starting target molecule.

Preparation of Templates to be Sequenced

Suitable templates for sequencing using the method of the invention canbe prepared by solid-phase nucleic acid amplification to produce nucleicacid colonies. The templates to be amplified will generally compriseunknown regions flanked by known ends, for example prepared according tomethods described in application WO07052006, whose contents areincorporated herein by reference in their entirety. For example, thetemplates may derive from a sample of genomic DNA, or from a cDNAlibrary. The amplification can be done using procedures analogous tothose described in WO 98/44151, WO 00/18957, WO0206456 or WO07107710,the contents of which are incorporated herein in their entirety byreference.

The templates for amplification may be prepared to contain a tagsequence, for example as shown in FIG. 12. The use of a tag sequence,for example as described in application WO05068656, whose contents areincorporated herein by reference in their entirety, allows multipledifferent samples to be analysed in the same sequencing run whilstpreserving the identity of each sample. FIG. 12 shows the tag that isread at the end of the first read, but the tag can equally be read atthe end of the second read, for example using a sequencing primercomplementary to the strand marked P7. The invention is not limited totwo reads per cluster, three or more reads per cluster are obtainablesimply by dehybridising a first extended sequencing primer, andrehybridising a second primer before or after the clusterrepopulation/strand resynthesis step. Methods of preparing suitablesamples for indexing are described in copending application U.S.60/899221 filed Feb. 1 2007.

For amplification to proceed, a mixture of at least two amplificationprimers is immobilised or “grafted” onto the surface of a suitable solidsupport.

The amplification primers are oligonucleotide molecules having thefollowing structures:

-   Forward primer: A-L-X-S1-   Reverse primer: A-L-Y-S2

Wherein A represents a moiety which allows attachment to the solidsupport, L is an optional linker or spacer moiety, X and Y are optionalcleavage sites as described above to allow subsequent removal of one orother of the strands from the surface and S1 and S2 are polynucleotidesequences which permit amplification of a template nucleic acid moleculecomprising the target double-stranded polynucleotide.

The mixture of primers will generally comprise substantially equalamounts the forward and reverse primers.

L represents a linker which may be included but is not strictlynecessary. The linker may be a carbon-containing chain such as those offormula (CH₂)_(n) wherein “n” is from 1 to about 1500, for example lessthan about 1000, preferably less than 100, e.g. from 2-50, particularly5-25. However, a variety of other linkers may be employed with the onlyrestriction placed on their structures being that the linkers are stableunder conditions under which the polynucleotides are intended to be usedsubsequently, e.g. conditions used in DNA amplification and sequencing.

Linkers which do not consist of only carbon atoms may also be used. Suchlinkers include polyethylene glycol (PEG) having a general formula of(CH₂—CH₂—O)_(m), wherein m is from about 1 to 600, preferably less thanabout 500.

Linkers formed primarily from chains of carbon atoms and from PEG may bemodified so as to contain functional groups which interrupt the chains.Examples of such groups include ketones, esters, amines, amides, ethers,thioethers, sulfoxides, and sulfones. Separately or in combination withthe presence of such functional groups may be employed alkene, alkyne,aromatic or heteroaromatic moieties, or cyclic aliphatic moieties (e.g.cyclohexyl). Cyclohexyl or phenyl rings may, for example, be connectedto a PEG or (CH₂)_(n) chain through their 1- and 4-positions.

As an alternative to the linkers described above, which are primarilybased on linear chains of saturated carbon atoms, optionally interruptedwith unsaturated carbon atoms or heteroatoms, other linkers may beenvisaged which are based on nucleic acids or monosaccharide units (e.g.dextrose). It is also within the scope of this invention to utilisepeptides as linkers.

In a further embodiment, a linker may comprise one or more nucleotideswhich form part of the amplification primer but which do not participatein any reaction carried out on or with the primer (e.g., a hybridisationor amplification reaction). Such nucleotides may also be referred toherein as “spacer” polynucleotides. Typically from 1 to 20, morepreferably from 1 to 15 or from 1 to 10, and more particularly 2, 3, 4,5, 6, 7, 8, 9 or 10 spacer nucleotides may be included. In a particularembodiment, the primer includes 10 spacer nucleotides. PolyT spacers maybe used in some embodiments, although other nucleotides and combinationsthereof can also be used. In a particular embodiment, the primer mayinclude 10T spacer nucleotides.

The one or more spacer nucleotides function to space the portion of theprimer required to hybridise to a target and direct amplification awayfrom the site of attachment to the solid support (i.e., S1 or S2). Theinclusion of spacer nucleotides at the 5′ end can markedly improve theperformance of hybridisation of complementary polynucleotides to regionS1 or S2. In a more particular embodiment the polynucleotide includes10T spacer nucleotides and a 5′ phosphorothioate group for attachment tothe solid support (moiety A), although other attachment moieties may beused as discussed below.

Sequences S1 and S2 in the forward and reverse primers arepolynucleotide sequences which, in combination, direct amplification ofa template by solid-phase bridging amplification reaction. The templateto be amplified must itself comprise (when viewed as a single strand) atthe 3′ end a sequence capable of hybridising to sequence S1 in theforward primers and at the 5′ end a sequence the complement of which iscapable of hybridising to sequence S2 the reverse primer.

The precise nature of sequences S1 and S2 in the forward and reverseprimer oligonucleotides will be dependent on the nature of the templateit is intended to amplify. S1 and S2 must be capable of hybridising tocognate sequences on complementary strands of the template to beamplified. The term “hybridisation” encompasses sequence-specificbinding between primer and template. Binding of a primer to its cognatesequence in the template should occur under typical conditions used forprimer-template annealing in standard PCR. Typically hybridisationconditions are 5×SSC at 40° C., following an initial denaturation step.It is not essential for hybridisation that sequences S1 and S2 beexactly complementary to their cognate sequences in the template to beamplified, although this is preferred.

S1 and S2 may be of different or identical sequence and will typicallybe around 20-30 nucleotides in length. The primers can include naturaland non-natural DNA bases, also ribonucleotides or any combinationthereof, and may also include non-natural backbone linkages such asdisulphides or phosphorothioates.

Cleavage site X and/or Y may fall within sequence S1 or S2, or if thelinker L is itself a polynucleotide cleavage they may form part oflinker region L. In other embodiments the cleavage site may be formed atthe junction of sequences L and S1 or L and S2, or at the junctionbetween moiety A and linker L (if present) or between moiety A andsequence S1 or S2 (if L not present).

Moiety A may be any chemical moiety which permits immobilisation of anoligonucleotide primer on a solid support. The surface of the solidsupport may itself be functionalised to permit attachment of theprimers. Any suitable covalent or non-covalent attachment means may beused, of which many are known in the art.

By way of example, biotinylated albumins (BSA) can form a stableattachment of biotin groups by physisorption of the protein ontosurfaces. Covalent modification can also be performed using silanes,which have been used to attach molecules to a solid support, usually aglass slide. By way of example, a mixture of tetraethoxysilane andtriethoxy-bromoacetamidopropyl-silane (e.g. in a ratio of 1:100) can beused to prepare functionalised glass slides which permit attachment ofmolecules such as nucleic acids including a thiophosphate orphosphorothioate functionality. Biotin molecules can be attached tosurfaces using appropriately reactive species such asbiotin-PEG-succinimidyl ester which reacts with an amino surface. Amixture of amplification primers may then be brought into contact withthe functionalised solid support.

In alternative embodiments functionalised polyacrylamide hydrogels maybe used to attach primers wherein moiety A is a sulfur-containingnucleophilic group.

Examples of appropriate sulfur nucleophile-containing polynucleotidesare disclosed in Zhao et al (Nucleic Acids Research, 2001, 29(4),955-959) and Pirrung et al (Langmuir, 2000, 16, 2185-2191) and include,for example, simple thiols, thiophosphates and thiophosphoramidates. Inparticular embodiments, hydrogels are formed from a mixture of (i) afirst comonomer which is acrylamide, methacrylamide, hydroxyethylmethacrylate or N-vinyl pyrrolidinone; and

(ii) a second comonomer which is a functionalised acrylamide or acrylateof formula (I):

H₂C═C(H)—C(═O)-A-B-C   (I);

or a methacrylate or methacrylamide of formula (II):

or H₂C═C(CH₃)—C(═O)-A-B-C-   (II)

(wherein:

A is NR or O, wherein R is hydrogen or an optionally substitutedsaturated hydrocarbyl group comprising 1 to 5 carbon atoms;

—B— is an optionally substituted alkylene biradical of formula—(CH₂)_(n)— wherein n is an integer from 1 to 50; and wherein n=2 ormore, one or more optionally substituted ethylene biradicals —CH₂CH₂— ofsaid alkylene biradical may be independently replaced by ethenylene andethynylene moieties; and wherein n=1 or more, one or more methylenebiradicals —CH₂— may be replaced independently with an optionallysubstituted mono- or polycyclic hydrocarbon biradical comprising from 4to 50 carbon atoms, or a corresponding heteromonocyclic orheteropolycyclic biradical wherein at least 1 CH₂ or CH₂ is substitutedby an oxygen sulfur or nitrogen atom or an NH group; and

C is a group for reaction with a compound (to bind the compoundcovalently to the hydrogel) to form a polymerised product. In aparticular embodiment, the hydrogel is formed by co-polymerisation ofacrylamide and N-(5-bromoacetamidylpentyl)acrylamide (BRAPA), asdescribed in application WO05065814, the contents of which areincorporated herein by reference in their entirity.

The term “solid support”, as used herein, refers to the material towhich the polynucleotides molecules are attached. Suitable solidsupports are available commercially, and will be apparent to the skilledperson. The supports can be manufactured from materials such as glass,ceramics, silica and silicon. Supports with a gold surface may also beused. The supports usually comprise a flat (planar) surface, or at leasta structure in which the polynucleotides to be interrogated are inapproximately the same plane. Alternatively, the solid support can benon-planar, e.g., a microbead. Any suitable size may be used. Forexample, the supports might be on the order of 1-10 cm in eachdirection.

For the grafting reaction to proceed a mixture of the amplificationprimers is applied to a (suitable functionalised) solid support underconditions which permit reaction between moiety A and the support. Theresult of the grafting reaction is a substantially even distribution ofthe primers over the solid support.

In certain embodiments the template to be amplified may be grafted ontothe solid support together with the amplification primers in a singlegrafting reaction. This can be achieved by adding template moleculesincluding moiety A at the 5′ end to the mixture of primers to form aprimer-template mixture. This mixture is then grafted onto the solidsupport in a single step. Amplification may then proceed using theimmobilised template and primers in a reaction analogous to thatdescribed in WO 00/18957. The first step in such a reaction will behybridisation between surface-bound templates and surface-boundamplification primers.

If the mixture of primers only is grafted onto the solid support and thetemplate to be amplified is present in free solution, the amplificationreaction may proceed substantially as described in WO 98/44151. Briefly,following attachment of the primers, the solid support is contacted withthe template to be amplified under conditions which permit hybridisationbetween the template and the immobilised primers. The template isusually added in free solution under suitable hybridisation conditions,which will be apparent to the skilled reader. Typically hybridisationconditions are, for example, 5×SSC at 40° C., following an initialdenaturation step. Solid-phase amplification can then proceed, the firststep of the amplification being a primer extension step in whichnucleotides are added to the 3′ end of the immobilised primer hybridisedto the template to produce a fully extended complementary strand. Thiscomplementary strand will thus include at its 3′ end a sequence which iscapable of binding to the second primer molecule immobilised on thesolid support. Further rounds of amplification lead to the formation ofclusters or colonies of template molecules bound to the solid support.

Sequences S1 and S2 in the amplification primers may be specific for aparticular target nucleic acid that it is desired to amplify, but inother embodiments sequences S1 and S2 may be “universal” primersequences which enable amplification of any target nucleic acid of knownor unknown sequence which has been modified to enable amplification withthe universal primers.

Suitable templates to be amplified with universal primers may beprepared by modifying target double-stranded polynucleotides by additionof known adaptor sequences to the 5′ and 3′ ends of the target nucleicacid molecules to be amplified. The target molecules themselves may beany double-stranded molecules it is desired to sequence (e.g., randomfragments of human genomic DNA). The adaptor sequences enableamplification of these molecules on a solid support to form clustersusing forward and reverse primers having the general structure describedabove, wherein sequences S1 and S2 are universal primer sequences.

The adaptors are typically short oligonucleotides that may besynthesised by conventional means. The adaptors may be attached to the5′ and 3′ ends of target nucleic acid fragments by a variety of means(e.g. subcloning, ligation. etc). More specifically, two differentadaptor sequences are attached to a target nucleic acid molecule to beamplified such that one adaptor is attached at one end of the targetnucleic acid molecule and another adaptor is attached at the other endof the target nucleic acid molecule. The resultant construct comprisinga target nucleic acid sequence flanked by adaptors may be referred toherein as a “template nucleic acid construct”. Suitable methods ofsample preparation for use in the method described herein are detailedin application number U.S. Ser. No. 11/486953.

The target double-stranded polynucleotides may advantageously besize-fractionated prior to modification with the adaptor sequences.

The adaptors contain sequences which permit nucleic acid amplificationusing the amplification primer molecules immobilised on the solidsupport. These sequences in the adaptors may be referred to herein as“primer binding sequences”. In order to act as a template for nucleicacid amplification, a single strand of the template construct mustcontain a sequence which is complementary to sequence S1 in the forwardamplification primers (such that the forward primer molecule can bindand prime synthesis of a complementary strand) and a sequence whichcorresponds to sequence S2 in the reverse amplification primer molecules(such that the reverse primer molecule can bind to the complementarystrand). The sequences in the adaptors which permit hybridisation toprimer molecules will typically be around 20-30 nucleotides in length,although the invention is not limited to sequences of this length.

The precise identity of sequences S1 and S2 in the amplificationprimers, and hence the cognate sequences in the adaptors, are generallynot material to the invention, as long as the primer molecules are ableto interact with the amplification sequences in order to direct bridgingamplification. The criteria for design of primers are generally wellknown to those of ordinary skill in the art.

Solid-phase amplification by either the method analogous to that of WO98/44151 or that of WO 00/18957 will result in production of an array ofcolonies of “bridged” amplification products. Both strands of theamplification product will be immobilised on the solid support at ornear the 5′ end, this attachment being derived from the originalattachment of the amplification primers. Typically the amplificationproducts within each colony will be derived from amplification of asingle target molecule.

Amplification methods suitable for the present invention include boththermocycling and isothermal amplifications. In a particular embodimentof the invention, isothermal amplification conditions, as described inapplication number W007107710, are used. The lower temperatures used inisothermal amplifications, which simply involve cycles of bufferexchange to change between extension and denaturing conditions, areadvantageous due to a higher retention of surface bound molecules, andtherefore brighter clusters (i.e, clusters having greater fluorescenceintensity following incorporation of a fluorescently labellednucleotide).

The utility of the sequencing method of the invention is not limited tosequencing of templates produced by an amplification reaction, althoughthis is preferred. The method may be applied to sequencing ofdouble-stranded templates immobilised on a support by any other meansamenable to repeated cycles of hybridisation and sequencing.

The invention will be further understood with reference to the followingnon-limiting experimental examples:

Examples

The following are examples of general techniques which may be applied incarrying out the method of the invention.

Example 1 Cluster Preparation Using the Detailed Invention AcrylamideCoating of Glass Chips

The solid supports used are typically 8-channel glass chips such asthose provided by Silex Microsystems (Sweden). However, the experimentalconditions and procedures are readily applicable to other solidsupports.

Chips were washed as follows: neat Decon for 30 min, milliQ H₂O for 30min, NaOH 1N for 15 min, milliQ H₂O for 30 min, HCl 0.1N for 15 min,milliQ H₂O for 30 min.

Polymer Solution Preparation:

For 10 ml of 2% polymerisation mix.

-   -   10 ml of 2% solution of acrylamide in milliQ H₂O    -   165 μl of a 100 mg/ml N-(5-bromoacetamidylpentyl)acrylamide        (BRAPA) solution in DMF (23.5 mg in 235 μl DMF)    -   11.5 μl of TEMED    -   100 μl of a 50 mg/ml solution of potassium persulfate in milliQ        H₂O (20 mg in 400 μl H₂O)

The 10 ml solution of acrylamide was first degassed with argon for 15min. The solutions of BRAPA, TEMED and potassium persulfate weresuccessively added to the acrylamide solution. The mixture was thenquickly vortexed and immediately used. Polymerization was then carriedout for 1 h 30 min at RT. Afterwards the channels were washed withmilliQ H₂O for 30 min and filled with 0.1 M potassium phosphate bufferfor storage until required.

Example 2 Synthesis of N-(5-bromoacetamidylpentyl)acrylamide (BRAPA) (1)

N-Boc-1,5-diaminopentane toluene sulfonic acid was obtained fromNovabiochem. The bromoacetyl chloride and acryloyl chloride wereobtained from Fluka. All other reagents were Aldrich products.

To a stirred suspension of N-Boc-1,5-diaminopentane toluene sulfonicacid (5.2 g, 13.88 mmol) and triethylamine (4.83 ml, 2.5 eq) in THF (120ml) at 0° C. was added acryloyl chloride (1.13 ml, 1 eq) through apressure equalized dropping funnel over a one hour period. The reactionmixture was then stirred at room temperature and the progress of thereaction checked by TLC (petroleum ether:ethyl acetate 1:1). After twohours, the salts formed during the reaction were filtered off and thefiltrate evaporated to dryness. The residue was purified by flashchromatography (neat petroleum ether followed by a gradient of ethylacetate up to 60%) to yield 2.56 g (9.98 mmol, 71%) of product 2 as abeige solid. ¹H NMR (400 MHz, d₆-DMSO): 1.20-1.22 (m, 2H, CH₂),1.29-1.43 (m, 13H, tBu, 2×CH₂), 2.86 (q, 2H, J=6.8 Hz and 12.9 Hz, CH₂),3.07 (q, 2H, J=6.8 Hz and 12.9 Hz, CH₂), 5.53 (dd, 1H, J=2.3 Hz and 10.1Hz, CH), 6.05 (dd, 1H, J=2.3 Hz and 17.2 Hz, CH), 6.20 (dd, 1H, J=10.1Hz and 17.2 Hz, CH), 6.77 (t, 1H, J=5.3 Hz, NH), 8.04 (bs, 1H, NH). Mass(electrospray+) calculated for C₁₃H₂₄N₂O₃ 256, found 279 (256+Na⁺).

Product 2 (2.56 g, 10 mmol) was dissolved in trifluoroaceticacid:dichloromethane (1:9, 100 ml) and stirred at room temperature. Theprogress of the reaction was monitored by TLC (dichloromethane:methanol9:1). On completion, the reaction mixture was evaporated to dryness, theresidue co-evaporated three times with toluene and then purified byflash chromatography (neat dichloromethane followed by a gradient ofmethanol up to 20%). Product 3 was obtained as a white powder (2.43 g, 9mmol, 90%). ¹H NMR (400 MHz, D₂O): 1.29-1.40 (m, 2H, CH₂), 1.52 (quint.,2H, J=7.1 Hz, CH₂), 1.61 (quint., 2H, J=7.7 Hz, CH₂), 2.92 (t, 2H, J=7.6Hz, CH₂), 3.21 (t, 2H, J=6.8 Hz, CH₂), 5.68 (dd, 1H, J=1.5 Hz and 10.1Hz, CH), 6.10 (dd, 1H, J=1.5 Hz and 17.2 Hz, CH), 6.20 (dd, 1H, J=10.1Hz and 17.2 Hz, CH). Mass (electrospray+) calculated for C₈H₁₆N₂O 156,found 179 (156+Na⁺).

To a suspension of product 3 (6.12 g, 22.64 mmol) and triethylamine(6.94 ml, 2.2 eq) in THF (120 ml) was added bromoacetyl chloride (2.07ml, 1.1 eq), through a pressure equalized dropping funnel, over a onehour period and at −60° C. (cardice and isopropanol bath in a dewar).The reaction mixture was then stirred at room temperature overnight andthe completion of the reaction was checked by TLC(dichloromethane:methanol 9:1) the following day. The salts formedduring the reaction were filtered off and the reaction mixtureevaporated to dryness. The residue was purified by chromatography (neatdichloromethane followed by a gradient of methanol up to 5%). 3.2 g(11.55 mmol, 51%) of the product 1 (BRAPA) were obtained as a whitepowder. A further recrystallization performed in petroleum ether:ethylacetate gave 3 g of the product 1. ¹H NMR (400 MHz, d₆-DMSO): 1.21-1.30(m, 2H, CH₂), 1.34-1.48 (m, 4H, 2×CH₂), 3.02-3.12 (m, 4H, 2×CH₂), 3.81(s, 2H, CH₂), 5.56 (d, 1H, J=9.85 Hz, CH), 6.07 (d, 1H, J=16.9 Hz, CH),6.20 (dd, 1H, J=10.1 Hz and 16.9 Hz, CH), 8.07 (bs, 1H, NH), 8.27 (bs,1H, NH). Mass (electrospray+) calculated for C₁₀H₁₇BrN₂O₂ 276 or 278,found 279 (278+H⁺), 299 (276+Na⁺).

Example 3 Grafting Primers onto Surface of SFA Coated Chip

An SFA coated chip is placed onto a modified MJ-Research thermocyclerand attached to a peristaltic pump. Grafting mix consisting of 0.5 μM ofa forward primer and 0.5 μM of a reverse primer in 10 mM phosphatebuffer (pH 7.0) is pumped into the channels of the chip at a flow rateof 60 μl/min for 75 s at 20° C. The thermocycler is then heated to 51.6°C., and the chip is incubated at this temperature for 1 hour. Duringthis time, the grafting mix undergoes 18 cycles of pumping: grafting mixis pumped in at 15 μl/min for 20 s, then the solution is pumped back andforth (5 s forward at 15 μl/min, then 5 s backward at 15 μl/min) for 180s. After 18 cycles of pumping, the chip is washed by pumping in5×SSC/5mM EDTA at 15 μl/min for 300 s at 51.6° C. The thermocycler isthen cooled to 20° C.

The primers are typically 5′-phosphorothioate oligonucleotidesincorporating any specific sequences or modifications required forcleavage. Their sequences and suppliers vary according to the experimentfor which they are to be used, and in this case were complementary tothe 5′-ends of the template duplex. The DNA sequence used in thisprocess was a single monotemplate sequence of 240 bases, with endscomplementary to the grafted primers. The full sequence of the templateduplex is shown in FIG. 6. The duplex DNA was denatured using sodiumhydroxide treatment followed by snap dilution as described.

For some of the experiments detailed, the amplified clusters contained adiol linkage in one of the grafted primers. Diol linkages can beintroduced by including a suitable linkage into one of the primers usedfor solid-phase amplification. Synthesis of the diol phosphoramidite isdescribed in Example 4 below. Products containing such diol linkages canbe cleaved using periodate and propanolamine as described, and theresulting single stranded polynucleotides hybridised as described.

The grafted primers contain a sequence of T bases at the 5′-end to actas a spacer group to aid linearisation and hybridization. The sequencesof the two primers grafted to the chip are designed to enable theappropriate treatment to release the free 3′-hydroxyl moiety, andcontain either U base as follows:

P5diol: 5′PS-TTTTTTTTTT-diol-AATGATACGGCGACCACCGA P7GAU:5′PS-TTTTTTTTTTCAAGCAGAAGACGGCATACGAGAU

The same primers can enable cutting with BstNB1, assuming the samples tobe amplified contain the relevant recognition site as follows:

5′ . . . G A G T C N N N N▾N . . . 3′ 3′. . . C T C A G N N N N N . . . 5′

To enable the cutting with the remote cutting restriction enzyme Mmel;the ends of both the grafting primers contained the following sequence:

5′ . . . TCCGAC-3′

To enable the cutting with the remote cutting restriction enzyme EcoP15;the ends of both the grafting primers contained the following sequence:

5′ . . . CAGCAG-3′

The design of the primers is shown in FIGS. 5 and 6

Example 4 Preparation of Diol-Phosphoramidite for DNA Coupling

Step 1:

1,6-Hexanediol (Sigma Aldrich 99%) (14.6 g, 124 mmol),N,N-diisopropylethylamine (Hünig's base; Sigma Aldrich; redistilled)(21.6 mL, 124 mmol) is dissolved in anhydrous DCM/DMF (250/50 mL) underN₂. The solution is cooled to 0° C. and the first portion of4,4′-dimethoxytrityl chloride (DMTr-Cl; Sigma-Aldrich 95%) (10.5 g, 31mmol) is added. The reaction mixture is then warmed to room temperature.After stirring for 1 h, the reaction mixture is cooled to 0° C. againand the second portion of DMTr-Cl (10.5 g, 31 mmol) is added and thenallowed to stir at room temperature for other 2 hours. TLC (EtOAc:petroleum ether 4:6) analysis indicates ca. 95% consumption of startingmaterial derivative (DMTr-OH). The reaction is concentrated underreduced pressure and Aq. NaHCO₃ (sat.) solution (500 mL) is poured intothe residue. The resulting mixture is extracted with petroleumether/EtOAc (2:1) (3×1000 mL). The combined organic layers are driedover MgSO₄, and concentrated under vacuum. The residue is co-evaporatedwith xylene (2×100 mL) to remove DMF. The reaction mixture, ispre-absorbed on silica gel and subjected to flash chromatography usingsolvents containing 1% Et₃N petroleum ether to petroleum ether/EtOAc(7:3) as eluent. The yield of pale yellow oil is 16.58 g, 64%, with afurther 7.8 g (17%) of the bis-tritylated by-product.

TLC: R_(f): 0.35 (diol-1); R_(f): 0.7 (bis-tritylated by-product)(petroleum ether/EtOAc 6:4).

¹H NMR (400 MHz, CDCl₃): δ 1.32-1.44 (m, 4H, 2×CH₂), 1.54-1.68 (m, 4H,2×CH₂), 3.06 (t, J=6.6 Hz, 2H, CH₂O), 3.62-3.68 (m, 2H, CH₂OH), 3.81 (s,6H, 2×MeO), 6.83-6.85 (m, 4H, Ph), 7.24-7.35 (m, 7H, Ph), 7.45-7.47 (m,2H, Ph).

Step 2:

To a solution of Diol-1 (16.6 g, 39.5 mmol) in anhydrous DCM (200 mL),tetrapropylammonium perruthenate (TPAP; Sigma Aldrich 97%) (277 mg, 0.79mmol) is added under N₂ atmosphere. The solution is cooled to 0° C. andN-methylmopholine N-oxide (Sigma Aldrich 97%) (2.7 g, 23 mmol) is added.The reaction is warmed to room temperature. After 1 hour, the otherthree portions of N-methylmopholine N-oxide (3×2.0 g, 51.2 mmol) areadded within a period of four hours. TLC (EtOAc: petroleum ether 4:6)indicates the reaction goes to completion. The reaction is quenched withaq. NaHCO₃ (sat.) (1000 mL) and extracted to CH₂Cl₂ (4×1000 mL). Thecombined organic layers are dried over MgSO₄. The solution isconcentrated under reduced pressure. Diol-3, 9.9 g, 60%, is isolated byflash chromatography using solvents containing 1% Et₃N from petroleumether to petroleum ether/EtOAc (6:4) as eluent, as a pale yellow oil.

TLC: R_(f): 0.7 (petroleum ether/EtOAc 6:4).

¹H NMR (400MHz, CDCl₃): δ 1.30-1.37 (m, 2H, CH₂), 1.48-1.57 (m, 4H,2×CH₂), 2.34 (td, J=1.7 and 7.4 Hz, 2H, CH₂CHO), 2.97 (s, 2H, CH₂O),3.72 (s, 6H, 2×MeO), 6.73-6.76 (m, 4H, Ph), 7.10-7.26 (m, 7H, Ph),7.34-7.36 (m, 2H, Ph), 9.67 (t, J=1.7, 1H, CHO).

Step 3:

A solution of triphenylphosphine (Sigma-Aldrich 99%, ReagentPlus™).(39.3 g, 150 mmol) and 4-bromobutyl acetate (Sigma-Aldrich)(26 mL, 180mmol) in anhydrous toluene (300 mL) is heated under reflux for 36 hoursunder N₂ in an oil-bath (140° C.). During the reflux, oil isprecipitated out. The reaction mixture is cooled to room temperature.TLC (petroleum ether/EtOAc 7:3) analysis of the toluene solution showedthat there is still triphenylphosphine (R_(f): 0.8) left. Thesupernatant is decanted into another round-bottomed flask andconcentrated down to the approximate volume of 30 mL. The solution isheated under reflux again for another 12 hours. The supernatant isdecanted. The portions of oil are combined together, dissolved in water(500 mL) and extracted with EtOAc (2×500 mL). The combined organiclayers are back-extracted with water (150 mL). Two lots of aqueouslayers are combined, evaporated under reduced pressure. The resultingresidue is co-evaporated with acetonitrile (2×100 mL) to give 78.4 g,95% yield of a pale yellow oil. NMR indicates that the product is pure,and is used for the next step reaction without further purification.

TLC: R_(f): 0.0 (petroleum ether/EtOAc 7:3).

¹H NMR (400 MHz, CDCl₃): δ 1.63-1.73 (m, 2H, CH₂), 1.94 (s, 3H, 2×CH₃),2.06-2.16 (m, 2H, CH₂), 3.97-4.05 (m, 2H, CH₂P), 4.11 (t, J=6.0, 2H,CH₂O), 7.69-7.95 (m, 15H, Ph).

³¹P NMR (162 MHz, CDCl₃): 25.9 ppm.

Mass spec details: LC-MS (Electrospray positive): (M⁺) 377.

Step 4:

Diol-2 (10.34 g, 22.7 mmol) is weighed into a round-bottomed flask anddissolved with DCM (20 mL). The solution is then evaporated underreduced pressure until it gives a white foam. The flask is thensubjected to high vacuum for 24 h. To this flask, anhydrous THF (180 mL)is added under N₂. The resulting suspension is cooled to −78° C. with anacetone-dry ice bath. With vigorous stirring, KOBu^(t) (3.3 g, 29.5mmol) is added under N₂. Slowly the colour of the suspension turnsorange, and white solids are gradually precipitated out. To thissuspension, a solution of diol-3 (dried at 60° C. under high vacuum for1 h before the reaction), (9.5 g, 22.7 mmol) in THF (50 mL) is addeddrop wise over half an hour. Acetone-dry ice bath is then removed. Thereaction mixture is slowly warmed to room temperature and stirred foranother hour. The colour of the reaction mixture turns yellow after theaddition of diol-3. The reaction mixture is concentrated down underreduced pressure. The resulting residue is partitioned between DCM (800mL) and aq. NaCl (sat.) (800 mL). The aqueous layer is extracted with anadditional DCM (2×800 mL). The organic extractions are combined, driedover MgSO₄, filtered, and evaporated under reduced pressure to giveyellow oil. The oil is dissolved in THF/MeOH (125/100 mL) and cooled to0° C. To this solution, NaOH (1M in H₂O, 25 mL) is added. After allowingthe reaction to stir for 1 hour, TLC analysis indicates full consumptionof starting material. The reaction mixture is neutralized with aceticacid (1.5 mL). The reaction mixture is concentrated down under reducedpressure. The resulting residue is partitioned between DCM (800 mL) andaq. NaHCO₃ (sat.) (800 mL). The aqueous layer is extracted withadditional DCM (2×800 mL). The organic extractions are combined, driedover MgSO₄, filtered, and evaporated to give a pale yellow oil. Diol-4,6.45 g, 60% is isolated by flash chromatography using solventscontaining 1% Et₃N from petroleum ether to petroleum ether/EtOAc (5:5)as eluent, as a light yellow oil.

TLC: R_(f)=0.45 (petroleum ether/EtOAc 6:4).

¹H NMR (400 MHz, CDCl₃) δ 1.24-1.32 (m, 4H, 2×CH₂), 1.54-1.57 (m, 4H,2×CH₂), 1.93-1.96 (m, 2H, CH₂), 2.02-2.07 (m, 2H, CH₂), 2.96 (t, J=6.6Hz, 2H, CH₂O), 3.54-3.59 (m, 2H, CH₂OH), 3.72 (s, 6H, 2×MeO), 5.29-5.32(m, 2H, 2×═CH), 6.73-6.77 (m, 4H, Ph), 7.11-7.27 (m, 7H, Ph), 7.36-7.38(m, 2H, Ph).

Step 5:

To a solution of Diol-4 (5.68 g, 12 mmol) and imidazole (Sigma Aldrich,99%), (1.63 g, 24 mmol) in anhydrous DMF (100 mL), t-butyldiphenylsilylchloride (Sigma Aldrich, 98%), (4.05 mL, 15.6 mmol) is added drop wiseunder N₂ atmosphere at room temperature. The reaction is stirred for 1hour. TLC (petroleum ether/EtOAc 8:2) indicates that the startingmaterial is fully consumed. A saturated aq. NaHCO₃ solution (500 mL) isadded to quench the reaction. The resulting mixture is extracted withpetroleum ether/EtOAc (2:1) (3×500 mL). The organic layers are combined,dried over MgSO₄, filtered, and evaporated to give a yellow oil. Diol-5,8.14 g, 95% is isolated by flash chromatography using solventscontaining 1% Et₃N from petroleum ether to petroleum ether/EtOAc (9:1)as eluent, as a colourless oil.

TLC: R_(f)=0.7 (petroleum ether:EtOAc 8:2).

¹H NMR (400 MHz, CDCl₃): δ 0.97 (s, 9H, 3×Me), 1.19-1.30 (m, 4H, 2×CH₂),1.48-1.55 (m, 4H, 2×CH₂), 1.91-1.95 (m, 2H, CH₂), 2.01-2.06 (m, 2H,CH₂), 2.95 (t, J=6.6 Hz, 2H, CH₂O), 3.58 (t, J=6.3 Hz, 2H, CH₂O), 3.70(s, 6H, 2×MeO), 5.24-5.27 (m, 2H, 2×═CH), 6.72-6.75 (m, 4H, Ph),7.11-7.37 (m, 15H, Ph), 7.57-7.60 (m, 4H, Ph).

Step 6:

A mixture of diol-5 (9.27 g, 13 mmol), AD-mix-α (Sigma Aldrich), (18.2g), methanesulfonamide (Sigma Aldrich, 97%), (1.23 g, 13 mmol), t-BuOH(65 mL) and water (65 mL) is stirred together vigorously at 55° C. for14 h. The TLC analysis (petroleum ether:EtOAc 6:4) indicates ca. 95%consumption of the starting material. The reaction mixture is cooled toroom temperature, treated with sodium sulfite (15.3 g, 12 mmol), thenfurther stirred for 30 min. A saturated aq. NaHCO₃ solution (500 mL) isadded to the reaction. The resulting mixture is extracted with EtOAc(3×500 mL). The organic layers are combined, dried over MgSO₄, filtered,and evaporated to give yellow oil. Diol-6, 7.96 g, 82%, is isolated byflash chromatography (silica gel, Fluka, 70-230 mesh) using solventscontaining 1% Et₃N from petroleum ether to petroleum ether/EtOAc (1:1)as elutant, as a white solid.

TLC: R_(f)=0.3 (petroleum ether:EtOAc 6:4).

¹H NMR (400 MHz, CDCl₃) δ 1.07 (s, 9H, 3×Me), 1.41-1.7 (m, 12H, 6×CH₂),1.94 (d, J=4.3 Hz, 1H, OH), 2.94-2.95 (m, 1H, OH), 3.06 (t, J=6.6 Hz,2H, CH₂O), 3.61-3.63 (m, 2H, 2×CHOH), 3.73 (t, J=5.6 Hz, 2H, CH₂O), 3.81(s, 6H, 2×MeO), 5.24-5.27 (m, 2H, 2×═CH), 6.82-6.85 (m, 4H, Ph),7.21-7.47 (m, 15H, Ph), 7.57-7.60 (m, 4H, Ph).

Step 7:

To a solution of diol-6 (7.96 g, 13 mmol) and DMAP (Sigma-AldrichReagentPlus™, 99%). (260 mg, 2.13 mmol) in a mixture of pyridine (15 mL)and DCM (30 mL), acetic anhydride (Fluka 99%), (2.5 mL, 26.68 mmol) isadded at room temperature. TLC analysis (petroleum ether:EtOAc 6:4)indicates full consumption of the starting material after 1 h. Thereaction is quenched by saturated aq. NaHCO₃ solution (500 mL). After 5min. the mixture is extracted with DCM (3×500 mL). The organic layersare combined, dried over MgSO₄, filtered, and evaporated. The residue isco-evaporated with toluene (2×100 mL). The resulting yellow oil issubjected to a plug of silica gel (50 g, Fluka, 70-230 mesh) to removeDMAP using eluents containing 0.1% Et₃N from petroleum ether topetroleum ether/EtOAc (7:3) (250 mL each). The combined fractions ofproduct are concentrated to dryness. The resulting colourless oil isdissolved in THF (100 mL) and treated with TBAF (Sigma-Aldrich; 5% wtwater), (1 M in THF, 15 mL) at 0° C. The reaction solution is slowlywarmed to room temperature and stirred for 2 hours. TLC analysis(petroleum ether:EtOAc 6:4) indicates that desilylation is completed.The volatile solvent (THF) is evaporated under reduced pressure at lowtemperature. A saturated aq. NaHCO₃ solution (500 mL) is added to theresidue. The resulting mixture is extracted with EtOAc (3×500 mL). Theorganic layers are combined, dried over MgSO₄, filtered, and evaporatedto give yellow oil. Diol-7, 4.2 g, 66%, is isolated by flashchromatography using solvents containing 1% Et₃N from petroleum ether topetroleum ether/EtOAc (1:1) as elutant, as a white solid.

TLC: R_(f)=0.45 (petroleum ether:EtOAc 1:1).

¹H NMR (400 MHz, CDCl₃) δ 1.29-1.33 (m, 4H, 2×CH₂), 1.47-1.63 (m, 8H,4×CH₂), 1.99, 2.01 (2s, 6H, 2 MeC(O)), 3.00 (t, J=6.5 Hz, 2H, CH₂O),3.60-3.64 (m, 2H, CH₂O), 3.75 (s, 6H, 2×MeO), 4.92-4.97 (m, 2H,2×CHOAc), 6.76-6.80 (m, 4H, Ph), 7.15-7.29 (m, 7H, Ph), 7.38-7.40 (m,2H, Ph).

Step 8:

To a solution of diol-7 (2.08 g, 3.5 mmol) and diisopropylethylamine(Sigma Aldrich), (1.53 ml, 8.75 mmol) in DCM (17 mL), 2-cyanoethylN,N-diisopropylchlorophosphor-amidite (1.0 g, 4.2 mmol) is added dropwise at room temperature under N₂. After stirring for 1 hour, TLCanalysis (petroleum ether:EtOAc 4:6) indicates the full consumption ofthe starting material. The solvent (THF) is concentrated under reducedpressure. The resulting residue is subjected to chromatography directly.Diol-8, 2.5 g, 90%, is isolated by flash chromatography using solventscontaining 1% Et₃N from petroleum ether to petroleum ether/EtOAc (1:1)as eluent, as a colourless syrup.

TLC: R_(f)=0.55 (petroleum ether:EtOAc 4:6).

¹H NMR (400 MHz, CDCl₃) δ 1.09, 1.10, 1.11, 1.12 (4 s, 12H, N(CHMe₂)₂),1.26-1.31 (m, 4H, 2×CH₂), 1.45-1.56 (m, 8H, 4×CH₂), 1.95, 1.969, 1.971,1.98 (4 s, 6H, 2 MeCO), 2.56 (t, J=6.5 Hz, 2H, CH₂CN), 2.95 (t, J=6.5Hz, 2H, CH₂O), 3.49-3.55 (m, 4H, CH₂O), 3.72 (s, 6H, 2×MeO), 4.89-4.92(m, 2H, 2×CHOAc), 6.74-6.76 (m, 4H, Ph), 7.13-7.25 (m, 7H, Ph),7.34-7.37 (m, 2H, Ph).

³¹P NMR (162 MHz, CDCl₃): 148.67, 148.69 ppm.

Example 5 Preparation of Clusters by Isothermal Amplification Step 1:Hybridisation and Amplification

The DNA sequence used in the amplification process is a singlemonotemplate sequence of 240 bases, with ends complementary to thegrafted primers. The full sequence of one strand of the template duplexis shown in FIG. 6. The duplex DNA (1 nM) is denatured using 0.1 Msodium hydroxide treatment followed by snap dilution to the desired0.2-2 pM ‘working concentration’ in ‘hybridization buffer’ (5×SSC/0.1%Tween).

Surface amplification was carried out by isothermal amplification usingan MJ Research thermocycler, coupled with an 8-way peristaltic pumpIsmatec IPC ISM931 equipped with Ismatec tubing (orange/yellow, 0.51 mmID). A schematic of the instrument is shown below.

The single stranded template is hybridised to the grafted primersimmediately prior to the amplification reaction, which thus begins withan initial primer extension step rather than template denaturation. Thehybridization procedure begins with a heating step in a stringent bufferto ensure complete denaturation prior to hybridisation. After thehybridization, which occurs during a 20 min slow cooling step, theflowcell was washed for 5 minutes with a wash buffer (0.3×SSC/0.1%Tween).

A typical amplification process is detailed in the following table,detailing the flow volumes per channel:

1. Template Hybridization and 1^(st) Extension

T Time Flow rate Pumped V Step Description (° C.) (sec) (μl/min) (μl) 1Pump Hybridization pre-mix 20 120 60 120 2 Pump Hybridization mix 98.5300 15 75 3 Remove bubbles 98.5 10 100  16.7 4 Stop flow and hold T 98.530 static 0 5 Slow cooling 98.5-40.2 19.5 min static 0 6 Pump washbuffer 40.2 300 15 75 7 Pump amplification pre-mix 40.2 200 15 50 8 Pumpamplification mix 40.2 75 60 75 9 First Extension 74 90 static 0 10 coolto room temperature 20 0 static 0

2. Isothermal Amplification

T Time Flow rate Pumped V Step Description (° C.) (sec) (μl/min) (μl)(1) Pump Formamide 60 75 60 75 This Pump Amplification 60 75 60 75sequence pre-mix 35 Pump Bst mix 60 95 60 95 times Stop flow and hold T60 180 static 0 2 Pump wash buffer 60 120 60 120

Hybridisation pre mix (buffer)=5×SSC/0.1% Tween

Hybridisation mix=0.1 M hydroxide DNA sample, diluted in hybridisationpre mix

Wash buffer=0.3×SSC/0.1% tween

Amplification pre mix=2 M betaine, 20 mM Tris, 10 mM Ammonium Sulfate, 2mM Magnesium sulfate, 0.1% Triton, 1.3% DMSO, pH 8.8

Amplification mix=2 M betaine, 20 mM Tris, 10 mM Ammonium Sulfate, 2 mMMagnesium sulfate, 0.1% Triton, 1.3% DMSO, pH 8.8 plus 200 μM dNTP's and25 units/mL of Taq polymerase (NEB Product ref MO273L)

Bst mix=2 M betaine, 20 mM Tris, 10 mM Ammonium Sulfate, 2 mM Magnesiumsulfate, 0.1% Triton, 1.3% DMSO, pH 8.8 plus 200 μM dNTP's and 80units/mL of Bst polymerase (NEB Product ref M0275L).

Step 2: Blocking Extendable 3′-OH Groups

To prepare the blocking pre-mix, 1530 μL of water and 170 μL of 10×blocking buffer (NEB buffer 4; product number B7004S) are mixed for afinal volume of 1700 μL. To prepare the blocking mix 1065.13 μL ofblocking pre-mix, 21.12 μL of 125 μM ddNTP mix, and 13.75 μL of TdTterminal transferase (NEB; part no M0252S) are mixed for a final volumeof 1100 μL.

To block the nucleic acid within the clusters formed in the flow cellchannels, the computer component of the instrumentation flows theappropriate blocking buffer through the flow cell, and controls thetemperature as shown in the exemplary embodiments below.

T Time Flow rate Pumped V Step Description (° C.) (sec) (μl/min) (μl) 1Pump Blocking 20 200 15 50 pre-mix 2 Pump Blocking mix 37.7 300 15 75 3Stop flow and 37.7  20 static 0 hold T 4 Cyclic pump 37.7 8 × 15/ 45Blocking mix and (20 + 180) static wait 5 Pump wash buffer 20 300 15 75

Example 6 Treatment of Clusters to Obtain a Free 3′-hydroxyl GroupMethod a: USER™

The amplified clusters were treated on the MJ platform to the followingconditions; flow with USER premix (10 mM KCl, 20 mM Tris-HCl (pH 8.8) 10mM (NH₄)₂SO₄, 2 mM MgSO4, 0.1% Triton-X 100) for 5 min at 38° C.; flowwith USER buffer (User premix plus 10 units/mL USER enzyme mix (NEB cat#M5505) for 5 mins and incubate for 45 min at 38° C.; wash in washbuffer for 5 mins.

The cleaved strands were treated with T7 exonuclease (100 units/mL inNEB buffer 4 (1×NEB4=50 mM potassium acetate, 20 mM Tris-acetate, 10 mMMagnesium acetate, 1mM DTT pH 7.9) for 30 mins at 30° C. and flushedwith wash buffer.

The strands were extended by ligation of SBS 5 ligation primer:

3′-GTAGCTGAGCCAAGTCGTCCTTACGGCTCTGGCT-PO4-5′

The primer (1 μM) in hybridization pre mix was flowed into the cell at60° C. and allow to hybridise for 5 mins. The cell was flushed with T4ligation buffer and cooled to 30° C. T4 DNA ligase was added, and thereaction held for 30 mins before flushing with wash buffer. The 3′-endof the hybridized ligated SBS 5 primer is available for subsequentextension reactions.

Method b: Nt.BstNBI

The amplified clusters were treated on the MJ platform with thefollowing conditions; the flow cells were washed with NEB buffer 3 at55° C. for 5 mins at 15 μl/min, then treated with 50 μl of Nt.BstNBI(1×NEB buffer 3; final enzyme conc 1000 units/mL). The cell wasincubated for 1 hr at 55° C. then lowered to 20° C. flushed with NEBbuffer 3 (5 min at 15 μl/min), then wash buffer (5 min at 15 μl/min).

The cleaved strands were treated with T7 exonuclease (100 units/mL inNEB buffer 4 (1×NEB4=50 mM potassium acetate, 20 mM Tris-acetate, 10 mMMagnesium acetate, 1 mM DTT pH 7.9) for 30 mins at 30° C. and flushedwith wash buffer.

Example 7 Sequencing from an Immobilized Primer

Sequencing of the clusters from the above illustrative protocol wascarried out using modified nucleotides prepared as described inInternational patent application WO 2004/018493, and labeled with fourspectrally distinct fluorophores, as described in U.S. application No.60/801270; filed May 18^(th) 2006. Sequencing of clusters is describedin more detail in patent WO06064199.

A mutant 9° N polymerase enzyme (an exo-variant including the triplemutation L408Y/Y409A/P410V and C223S) was used for the nucleotideincorporation steps.

Incorporation mix, Incorporation buffer (50 mM Tris-HCl pH 8.0, 6 mMMgSO4, 1 mM EDTA, 0.05% (v/v) Tween-20, 50 mM NaCl) plus 110 nM YAVexo-C223S, and 1 μM each of the four labeled modified nucleotides, wasapplied to the clustered templates, and heated to 45° C.

Templates were maintained at 45° C. for 30 min, cooled to 20° C. andwashed with Incorporation buffer, then with 5×SSC/0.05% Tween 20.Templates were then exposed to Imaging buffer (100 mM Tris pH7.0, 30 mMNaCl, 0.05% Tween 20, 50 mM sodium ascorbate, freshly dissolved).Templates were scanned in 4 colors at room temp. Templates were thenexposed to sequencing cycles of Cleavage and Incorporation as follows:

Cleavage: Prime with Cleavage buffer (0.1M Tris pH 7.4, 0.1M NaCl and0.05% Tween 20), 125 μL/channel; 60 μL/min.

Heat to 60° C.

Treat the clusters with Cleavage mix (100 mM TCEP in Cleavage buffer),75 μL/channel; 60 μL/min.

Wait for a total of 15 min in addition to pumping fresh cleavage mix, 25μL/channel; 60 μL/min, every 4 min.

Cool to 20° C.

Wash with Enzymology buffer.

Wash with 5×SSC/0.05% Tween 20.

Prime with Imaging buffer.

Scan in 4 colors at RT.

Incorporation: Prime with Incorporation buffer, 125 μL/channel; 60μL/min, Heat to 60° C.

Treat with Incorporation mix, 75 μL/channel; 60 μL/min.

Wait for a total of 15 min in addition to pumping fresh Incorporationmix, 25 μL/channel; 60 μL/min, every 4 min.

Cool to 20° C.

Wash with Incorporation buffer, 75 μL/channel; 60 μL/min.

Wash with 5×SSC/0.05% Tween 20, 75 μL/channel; 60 μL/min

Prime with imaging buffer, 100 μL/channel; 60 μL/min

Scan in 4 colors at RT.

Repeat the process of Incorporation and Cleavage for as many cycles asrequired.

Incorporated nucleotides were detected using a Total Internal Reflectionbased fluorescent CCD imaging apparatus described in “Systems andDevices for Sequence by Synthesis Analysis,” U.S. Ser. No. 60/788,248,filed Mar. 31, 2006.

A representative image showing two cycles of incorporation is shown inFIG. 7. The first cycle of incorporation (top right) shows an image with3271 clusters that have incorporated a G triphosphate according to themonotemplate sequence. The second cycle (bottom right), shows an imageof the second cycle where 3235 clusters have incorporated an Atriphosphate. The main picture shows the colocalised image, where 3058of the clusters are common to both images, showing that the clusters cango through cycles of sequencing from the immobilized primer.

Example 8 Extension with Unlabelled Nucleotides

After the final deblock step of the sequencing protocol, the array wastreated to a round of extension with four dNTP's and Bst polymerase,analogous to a cycle of extension in the cluster generation process. Thearray was treated as follows:

T Time Flow rate Pumped V Description (° C.) (sec) (μl/min) (μl) PumpAmplification pre-mix 60 75 60 75 Pump Bst mix 60 95 60 95 Stop flow andhold T 60 180 static 0 Pump wash buffer 60 120 60 120

Example 9 Treatment to Excise the Central Region of a Cluster

Clusters were treated with Mmel, followed by T4 polymerase to remove thedinucleotide overhangs, and T4 ligase to join the polished, blunt endsin a 36 base pair sequence. Identical protocols have been described insolution on circular vector constructs, (Nature Methods, 2, 105-111(2005)), and the methods were repeated on the amplified clusters, usingbuffer conditions and temperatures as recommended for the appropriateenzyme.

Example 10 Linearisation and Hybridization of a Sequencing Primer

To prepare the linearization mix 1429 uL of water, 64 mg of sodiumperiodate, 1500 uL of formamide, 60 uL of 1M Tris pH8, and 6011.4 uL of3-aminopropanol are mixed for a final volume of 3 mL. The periodate isfirst mixed with the water while the Tris is mixed with the formamide.The two solutions are then mixed together and the 3-aminopropanol isadded to that mixture.

To linearize the nucleic acid within the clusters formed within the flowcell channels, 300 μL per channel of linearisation mix is flowed in at15 μL/min at 20° C.; followed by 75 μL of water at the same flow rate.

To prepare the primer mix, 895.5 μL of hybridization buffer and 4.5 μlof sequencing primer (100 μM) are mixed to a final volume of 900 μL. Thesequence of the sequencing primer used in this reaction was:

5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC

To denature the nucleic acid within the clusters and to hybridize thesequencing primer, the following reagents are flowed through the cell:

T Time Flow rate Pumped V Step Description (° C.) (sec) (μl/min) (μl) 1Pump 0.1 M NaOH 20 300 15 75 2 Pump TE 20 300 15 75 3 Pump Primer mix 20300 15 75 4 Hold at 60 C. 60 900 0 0 5 Pump wash buffer 40.2 300 15 75

Example 11 Sequencing from a Non-Immobilised Primer

The sequencing process was carried out in exactly the same way asdescribed in Example 7. The second read can be aligned with the first,in the case of two reads, or the sequencing process was carried out for36 cycles in the case of Mmel treatment. This single read generated 18bases of information for each end of the original fragment.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

Example 12 Paired Reads from a Library of Fragments

The following experimental details describe the complete exposition ofone embodiment of the invention as described above.

The library was made using purified human BAC DNA (140k human chromosome6 insert cloned into a pTARBAC vector). The DNA was first prepared forligation to forked adaptors by: fragmentation of the DNA bynebulisation, end repair of the DNA ends to make them blunt-ended andphosphorylated, then the addition of a single ‘A’ nucleotide onto the 3′ends of the DNA fragments. The ligation reaction was performed with theprepared fragmented DNA and adaptors pre-formed by annealing ‘Oligo A’and ‘Oligo B’ (sequences given below). The product of the reaction wasisolated/purified from unligated adaptor by gel electrophoresis.Finally, the product of the ligation reaction was subjected to cycles ofPCR to selectively amplify ligated product that contained genomic DNAwith adaptor at both ends of the fragments.

Materials and Methods Step 1) Nebulization Materials:

-   -   0.5 ug/ul Human BAC DNA (140k human chromosome 6 insert cloned        into a pTARBAC vector)

Nebulization Buffer (53.1 ml glycerol, 42.1 ml water, 3.7 ml 1 M TrisHCl pH7.5, 1.1 ml 0.5 M EDTA)

-   -   TE    -   Nebulizers (Invitrogen, K7025-05)    -   PCR purification kit columns (Qiagen, 28104)

Procedure:

Mixed 10 μl (5 μg) of BAC DNA with 40 μl of TE and 700 μl ofnebulization buffer. Chilled DNA solutions were each fragmented in anebulizer on ice for 6 minutes under 32 pounds per square inch (psi) ofpressure. The recovered volumes were each purified with a Qiagen PCRpurification kit column and eluted in 30 μl of EB.

Step 2) End-Repair

Materials:

-   -   Nebulized DNA (from Step 1)    -   Water    -   T4 DNA ligase buffer with 10 mM ATP (10×) (NEB, B0202S)    -   dNTPs mix (10 mM each) (NEB, N0447S)    -   T4 DNA Polymerase (3U/ul) (NEB, M0203L)    -   E. coli DNA Pol I large fragment (Klenow) (5 U/ul) (NEB, M0210S)    -   T4 polynucleotide kinase (10 U/ul) (NEB, M0201L)    -   PCR purification kit columns (Qiagen, 28104)

Procedure:

End Repair Mix was Assembled as Follows:

Nebulized DNA  30 μl Water  45 μl T4 DNA ligase buffer with 10 mM ATP 10 μl dNTPs  4 μl T4 DNA pol  5 μl Klenow DNA pol  1 ul T4 PNK  5 ul100 μl total

The reaction was incubated for 30 mins at room temperature. The DNA waspurified on a Qiagen column, eluting in 30 μl EB.

Step 3) A-Tailing Reaction

Materials:

-   -   End repaired DNA (from Step 2)    -   Water    -   NEB buffer 2 (10×) (NEB, B7002S)    -   dATP (1 mM) (Amersham-Pharmacia, 272050)    -   Klenow fragment (3′ to 5′ exo minus) (5 U/μl) (NEB, M0212B)    -   Hot block or PCR machine    -   MinElute PCR purification kit column (Qiagen, 28004)

Procedure:

The following reaction mix was assembled:

End repaired DNA 30 μl Water  2 ul NEB buffer 2  5 μl dATP 10 μl Klenowfragment (3′ to 5′ exo minus)  3 μl 50 μl total

The reaction was incubated for 30 min at 37° C., then the DNA purifiedon a Qiagen MinElute column, eluting in 10 μl EB.

Step 4) Annealed Adaptors

Materials:

-   -   Oligo A: 5′ACACTCTTTCCCTACACGACGCTCTTCCGATCxT        (x=phosphorothioate bond)    -   Oligo B: 5′Phosphate-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG    -   5 0mM Tris/50mM NaCl pH7.0    -   PCR machine

Procedure:

The oligos were mixed together to a final concentration of 15 μM each,in 10 mM Tris/10 mM NaCl pH 7.0. The adaptor strands were annealed in aPCR machine programmed as follows: Ramp at 0.5° C./sec to 97.5° C.; Holdat 97.5° C. for 150 sec; then a step of 97.5° C. for 2 sec with atemperature drop of 0.1° C./cycle for 775 cycles.

Step 5) Ligation

Materials:

-   -   A-tailed genomic DNA (from Step 3)    -   Quick ligase buffer (2×) (NEB, B2200S)    -   Annealed adaptor (15 μM) (from 4.)    -   Quick Ligase (1 U/μl) (NEB, M2200L)    -   PCR purification kit columns (Qiagen, 28104)

Procedure:

Reaction mix was assembled as follows:

A-tailed genomic DNA 10 μl Quick ligase buffer 25 μl Annealed adaptor 10μl Quick Ligase  5 μl 50 μl total

The reaction was incubated for 15 min at room temperature, then the DNApurified on a Qiagen column, eluting in 30 μl Elution buffer (EB).

Step 6) Gel Purification

Materials:

-   -   Ligation reaction (from Step 5)    -   Agarose (Biorad, 161-3107)    -   TAE (50×)    -   Distilled water    -   Ethidium bromide (Sigma, E1510)    -   Loading buffer (4×) (50 mM Tris pH8, 40 mM EDTA, 40% w/v        sucrose)    -   Low molecular weight ladder (NEB, N3233L)    -   Gel trays and tank. Electrophoresis unit    -   Dark reader transilluminator (Clare Chemical Research, D195M)    -   Gel extraction kit columns (Qiagen, 28704)

Procedure:

The entire sample from the purified ligation reaction was loaded intoone lane of a 2% agarose gel containing ethidium bromide and run at 120Vfor 60 min. The gel was then viewed on a ‘White-light’ box and fragmentsfrom 120 bp to 170 bp excised and purified with a gel extraction column,eluting in 30 μl elution buffer (EB).

Step 7) Exonuclease I Treatment of PCR Primers Materials:

-   -   Exonuclease I (E. coli) (20 U/ul) (NEB, M0293S)    -   Exonuclease I Reaction Buffer (10×) (NEB, M0293S)    -   Water    -   DNA Primers with a phosphorothioate at the n-1 position    -   P6 Bio-Rad columns (Bio-Rad, 732-6221)

Procedure:

DNA Primers with a phosphorothioate at the n-1 position (5×85 μl of eachPrimer (approx 25 μM)) were aliquoted into Eppendorf tubes. 10 μl of10×Exonuclease I Reaction Buffer and 5 μl of Exonuclease I was added toeach tube. Each Eppendorf tube was placed in a rack and stored in anoven set at 37° C. for 16 hours. After 16 hr, the tubes were placed on ahotblock set at 80° C. for 2 minutes. Subsequently, the solutions fromthe Eppendorfs were passed through P6 Bio-Rad columns and spun in acentrifuge at 2000 rpm for 2 minutes. An extra 20 μl of H₂O was added tothe columns and the columns respun. The filtered solutions were placedinto a SpeedVac® and evaporated until each was at 20 μl, and thefractions combined. The pooled fractions were injected into a reversephase HPLC system, and the main peak was collected. The collectedfractions were evaporated to dryness in a SpeedVac®), 50 μl of water wasadded and the fraction was subjected again to evaporation to dryness.The resulting pellets were dissolved in 50 μl of water, pooled and a UVmeasurement taken to determine the concentration of the oligonucleotide.

Step 8) PCR

Materials:

-   -   Gel purified DNA (from Step 6)    -   Water    -   Phusion master mix (2×) (NEB, F-531L)    -   Exonuclease treated universal PCR primer 1 (25 uM): 5′        AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGA TCxT 3′,        where x=phosphorothioate bond (from Step 7)    -   Exonuclease treated universal PCR primer 2 (25 uM): 5′        CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTC CGATCxT,        where x=phosphorothioate bond (from Step 7)    -   PCR machine    -   PCR purification kit columns (Qiagen, #28104)

Procedure:

The PCR reaction was prepared as follows:

Gel purified BAC DNA  1 μl Phusion mastermix 25 μl Universal PCR primer1  1 μl Universal PCR primer 2  1 μl Water 22 μl 50 μl total

Thermocycling was carried out in a PCR machine under the followingconditions:

-   -   30 secs @ 98° C.    -   [10 sec@ 98° C., 30 sec @ 65° C., 30 sec @ 72° C.] 18 cycles    -   5 min @ 72° C.    -   Hold @ 4° C.

PCR products were purified on a Qiagen column, eluting in 30 μl EB. Theresulting DNA libraries were ready for amplification on a surfaceamplification platform.

Validation of Libraries by Conventional Sanger Sequencing

Four (4) μl of the libraries were cloned into a plasmid vector (ZeroBlunt TOPO PCR cloning kit, Invitrogen #K2800-20) and plated out onagar, according to the manufacturer's instructions. Colonies werepicked, mini-prepped and the cloned fragments sequenced by conventionalSanger sequencing.

16 Clones from BAC Library

1 (204 bp) Insert: E. coli 85 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGATTTCATTGCAGCCAAAGGCAAACTTTGGCTGCATCGTTTTACAGTCGCCATAAGCCTTTCCTCTGTTAAACCGCCTTCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCC GTCTTCTGCTTG2 (214 bp) Insert: BAC 95 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTATCAATATTGTGAAAATGACCATACTGCCAAAAAAAAACTACAAATTCAATGCAATTTTCATCAAAATACCATCATCATTCTTCACAATATTGATAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGAT CTCGTATGCCGTCTTCTGCTTG3 (215 bp) Insert: BAC 96 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTCACTCCTGGCAGAGGGACGTGTGACTAGCCATGGGCCCCTAGGTCTCCAGTTCCTGGGTAGCTTGTATTTTTGAACATCTCCTGTATATTAGTTAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGA TCTCGTATGCCGTCTTCTGCTTG4 (147 bp) Insert: BAC 28 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTGTAGTTGAGATCTGCCTTAGCAGCAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGC TTG5 (183 bp) Insert: BAC 64 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAACACATTTCAAAGTTTGGGGCCCTCCTCCTCCCCAAAAAACAAACCACAAAAAACAAACAAAAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG 6 (170 bp) Insert: BAC 59 bpGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGAATGCCTTTTATAGCATTTAATTTTTCCTAAGTATAATTACCAAATAAAAATTGTATAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG 7 (180 bp) Insert: BAC 61 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGGCCCGGGAGGAGTTTGCCGGGGAGGAGTGGGTTTGGAATCGGGGTTAAAGGAAAGAGAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG 8 (190 bp) Insert: BAC 73 bpTGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGATCTATTTCAAATGGACTGTAGATCTAAGTATAAAAGGTAAGAGAATAATTATTCTAGAAAGTAAATGTAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG9 (192 bp) Insert: BAC 74 bpAATGATACGGCGACCACCAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGGAGGCCAAGGTGGGTGGATCACCTGAGATCAGGAGTTCGAGACCAGCTGGCCAACATGATGAAACTCTGTCTAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG10 (185 bp) Insert: BAC 66 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACCATTGTAACCATTAATGTAGACTGCAATGATATGCACTATTTACAACCTTTTTTAAGACTCTAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG 11 (199 bp) Insert: BAC 80 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTTGAAGAGCTGGCAGTAGAAGATAAACAGGCTGGGGAAGAAGAGAAAGTGCTCAAGGAGAAGGAGCAGCAGCAGCAGCAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTT CTGCTTG12 (212 bp) Insert: BAC 93 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTATTCAACAAGTCTGTCTTTTCCAAGTGTCTTTAAAGACCAGAAATACCTGTTTTTAACACACAGGGTTGCAAAATTCAGAGGAGATTGGCAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCT CGTATGCCGTCTTCTGCTTG13 (247 bp) Insert: E. coli 128 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAGATGAGTGATGACGGCGCGCTGGAAGTTGCTCGTCGCGCTCGCGGTACGCCGCGCATTGCCAACCGTCTGCTGCGTCGAGTGCGTGATTTCGCCGAAGTGAAGCACGATGGCACCATCTCAAGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTT CTGCTTG14 (202 bp) Insert: BAC 83 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGGGTTGGTGGAACCCAGATGCCTCCCAGGATTGGTGGGCCCTGTGGCACTTGTACCTGCTGTTGCTGTTGCTGCTGCTGCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGT CTTCTGCTTG15 (166 bp) Insert: BAC 47 bpAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTATGATAAGGAGCAGGTTTACAGATCATAAGTGCAAAAGCGGGCGAGAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGAT CTCGTATGCCGTCTTCTGCTTG16 (147 bp) Insert: BAC 31 bpGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGATACTGTTGTAACCACCCAATTGGTTCAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGC TTG

These results confirm that the library preparation method produces alibrary of ‘sequenceable’ DNA templates. Each library contained aplurality of genomic inserts, each of which was flanked by the twoadaptors (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT andAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTT G),required for cluster formation and SBS sequencing. The insert DNA fromeach of the libraries aligned to the BAC reference, with a small amountof E. coli contamination.

Clusters were prepared from the above library as described in examples1-5 above.

The primers grafted to the surface had the following sequence:

P5diol: 5′ PS-TTTTTTTTTT-diol-AATGATACGGCGACCACCGA P7-TU:5-TTTTTTTTTUCAAGCAGAAGACGGCATACGA-OH P5-phosphate: 5′PS-TTTTTTTTTTAATGATACGGCGACCACCGA-PO4

The three primers were mixed at a 1:1:1 concentration, and used at 0.5μM concentration of each primer.

The clusters were linearised using periodate treatment as described inexample 10, and blocked with terminal transferase as described inexample 5, step 2.

The clusters were treated to hybridise a first sequencing primer, asdescribed above. The sequence of the sequencing primer was5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC

To denature the nucleic acid within the clusters and to hybridize thesequencing primer, the following reagents are flowed through the cell asshown in Table 4:

T Time Flow rate Pumped V Step Description (° C.) (sec) (μl/min) (μl) 1Pump 0.1 M NaOH 20 300 15 75 2 Pump TE 20 300 15 75 3 Pump Primer mix 20300 15 75 4 Hold at 60 C. 60 900 0 0 5 Pump wash buffer 40.2 300 15 75Sequencing from the Target Fragment was Performed Using Reagents andMethods Described Above in example 7.

Example 12a Obtaining a Second Read

Step 1: Deblocking the Phosphate Groups with T4 Polynucleotide Kinase

The clusters were treated as follows: Flow 1× Exchange reaction buffer(50 mM Imidazole-HCl pH 6.4; 12 mM MgCl₂; 1 mM 2-mercaptoethanol; 70 μMADP) at 15 μL/channel/min for 5 mins at 20° C. This was followed by PNKsolution (0.2 U/μL of PNK in 1× exchange reaction buffer) at 15μL/channel/min for 5 mins at 20° C. The flow cell was heated to 38° C.for 30 mins and allowed to cool. The channels were washed with washbuffer for 5 minutes, then 5×SSC for 5 minutes.

Step 2: Resynthesis of Clusters.

The clusters were treated to 15 cycles of isothermal amplification asdescribed in example 5; step 1

Step 3: Linearisation with USER

The clusters were treated as described in example 6 to cleave the uracilmoiety in the P7 primer. The clusters were blocked with terminaltransferase as described in example 5, step 2.

The clusters were treated to hybridise a first sequencing primer, asdescribed above. The sequence of the sequencing primer was5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC

To denature the nucleic acid within the clusters and to hybridize thesequencing primer, the following reagents are flowed through the cell asshown in Table 4:

T Time Flow rate Pumped V Step Description (° C.) (sec) (μl/min) (μl) 1Pump 0.1 M NaOH 20 300 15 75 2 Pump TE 20 300 15 75 3 Pump Primer mix 20300 15 75 4 Hold at 60 C. 60 900 0 0 5 Pump wash buffer 40.2 300 15 75Sequencing from the Target Fragment was Performed Using Reagents andMethods Described Above in example 7.

The data obtained from the first and second reads is shown in FIG. 8.Both reads clearly align against the BAC sequence, with only 4% of thereads deriving from the E. coli that contaminated the original sample.The average fragment size of the inserts was 80 base pairs, so for thetwo 25 base pair reads, the distance between the ends of the reads was30 base pairs on average. The majority of the clusters from the firstread (8208/8572) (96%) also gave a good read for the second read.

Example 13 Preparation and Paired End Sequencing Using Two ImmobilisedPrimers Modified with Uracil and 8-oxo G Respectively Step 1) GraftingPrimers Onto Surface of SFA Coated Chip

An SFA coated chip is placed onto a modified MJ-Research thermocyclerand attached to a peristaltic pump. Grafting mix consisting of 0.5 μM ofa forward primer and 0.5 μM of a reverse primer in 10 mM phosphatebuffer (pH 7.0) is pumped into the channels of the chip at a flow rateof 60 μl/minutes for 75 s at 20° C. The thermocycler is then heated to51.6° C., and the chip is incubated at this temperature for 1 hour.During this time, the grafting mix undergoes 18 cycles of pumping:grafting mix is pumped in at 15 μl/minutes for 20 s, then the solutionis pumped back and forth (5 s forward at 15 μl/minutes, then 5 sbackward at 15 μl/minutes) for 180 s. After 18 cycles of pumping, thechip is washed by pumping in 5×SSC/5 mM EDTA at 15 μl/minutes for 300 sat 51.6° C. The thermocycler is then cooled to 20° C.

The primers are typically 5′-phosphorothioate oligonucleotidesincorporating any specific sequences or modifications required forcleavage. Their sequences and suppliers vary according to the experimentfor which they are to be used, and in this case were complementary tothe 5′-ends of the template duplex. The DNA sequence used in thisprocess was the pool of the two libraries, which have ends complementaryto the grafted primers. The library mix was denatured using sodiumhydroxide treatment followed by snap dilution as described.

For some of the experiments detailed, the amplified clusters contained adiol linkage in one of the grafted primers. Diol linkages can beintroduced by including a suitable linkage into one of the primers usedfor solid-phase amplification. Synthesis of the diol phosphoramidite isdescribed in Example 4 below. Products containing such diol linkages canbe cleaved using periodate and propanolamine as described, and theresulting single stranded polynucleotides hybridised as described.

The grafted primers contain a sequence of T bases at the 5′-end to actas a spacer group to aid linearisation and hybridization. The sequencesof the three primers grafted to the chip are as follows:

Oligo A: 5′-PS-TTTTTTTTTTAATGATACGGCGACCACCGAUCT ACAC-3′ where U =2-deoxyuridine; Oligo B: 5′-PS-TTTTTTTTTTCAAGCAGAAGACGGCATACGAGoxoAT-3′, where Goxo = 8-oxoguanine).

Step 2) Preparation of Clusters by Isothermal Amplification:

The DNA sequence used in the amplification process is the mixture of thetwo libraries prepared in Example 1, which have ends complementary tothe grafted primers. The duplex DNA (1 nM) is denatured using 0.1 Msodium hydroxide treatment followed by snap dilution to the desired0.2-2 pM ‘working concentration’ in ‘hybridization buffer’ (5×SSC/0.1%Tween).

Surface amplification was carried out by isothermal amplification usinga commercially available Solexa/Illumina cluster station as described inPCT/US/2007/014649. The cluster station is essentially a hotplate and afluidics system for controlled delivery of reagents to a flow cell.

The single stranded template (denatured as indicated above) ishybridised to the grafted primers immediately prior to the amplificationreaction, which thus begins with an initial primer extension step ratherthan template denaturation. The hybridization procedure begins with aheating step in a stringent buffer to ensure complete denaturation priorto hybridisation. After the hybridization, which occurs during a 20minute slow cooling step, the flowcell was washed for 5 minutes with awash buffer (0.3×SSC/0.1% Tween).

During template hybridization and first extension, a number ofsolutions/buffers are typically employed, e.g., a solution comprisingthe DNA samples, a hybridization buffer (5×SSC/0.1% tween), a washbuffer (0.3×SSC/0.1% tween), a 2M sodium hydroxide solution, a clusterbuffer (200 mM Tris, 100 mM Ammonium Sulfate, 20 mM Magnesium sulfate,1% Triton, 1.3% DMSO, pH 8.8); an amplification additive (5 M betaine),DNA polymerase, and 10 mM dNTP mix.

To prepare the hybridization mixes, a 0.2 ml strip sample tube and thehybridization buffer are pre-chilled. Using 1.7 ml Eppendorf tube(s),the DNA template(s) are then diluted to 1 nM in buffer EB (Qiagen). 1 μLof 2 M NaOH is added to 19 μL of template, vortexed briefly andincubated for 5 minutes at room temperature to denature the DNA templateinto single strands. The denatured DNA is diluted to workingconcentration (0.2-2 pM) in pre-chilled hybridization buffer (e.g. for 1mL of 1 pM Hybridization mix, 1 μL of denatured DNA is diluted into 1 mLof pre-chilled hybridization buffer). The volume required depends on thenumber of channels used—at least 120 μL of hybridization mix per channelis optionally used. Thus, 1 mL of hybridization mix is enough for 8channels. The samples are vortexed briefly, spun down and aliquoted intothe pre-chilled 0.2 ml strip tubes (with no bubbles in the bottom of thetubes) and used immediately.

To prepare the Amplification pre-mix (of sufficient volume for the firstextension and 35 cycles of isothermal amplification), 35 mL of H₂0(milliQ), 7 mL of Cluster buffer (200 mM Tris, 100 mM Ammonium Sulfate,20 mM Magnesium sulfate, 1% Triton, 1.3% DMSO, pH 8.8), and 28 mL ofAmplification additive (5 M betaine solution) are mixed to achieve afinal volume of 70 mL.

To prepare the first extension Taq mix, 780 μL of Amplification pre-mix,16 μL of 10 mM dNTPs, and 4 μL of Taq DNA polymerase are mixed togetherfor a final volume of 800 μl.

A typical amplification process is detailed in the following table(Table 1), detailing the flow volumes per channel, controlledautomatically by the computer component of the invention.

TABLE 1 Template hybridization and first extension. T Time Flow ratePumped V Step Description (° C.) (sec) (μl/min) (μl) 1 PumpHybridization 20 120 60 120 pre-mix 2 Pump Hybridization mix 96 300 1575 3 Remove bubbles 96 6 100  10 4 Stop flow and hold T 96 30 static 0 5Slow cooling 96-40 1120 static 0 6 Pump wash buffer 40 300 15 75 7 Pumpamplification pre-mix 40 280 15 70 8 Pump amplification mix 40 95 60 959 First Extension 74 90 static 0 10 cool to room temperature 20 0 static0Isothermal Amplification at 60° C. using Formamide as Denaturant

The copied DNA can be isothermally amplified into clusters at 60° C.using formamide as a denaturant. The isothermal amplification (includingboth temperature control and reagent control) is overseen by thecomputer component. Table 2 gives outlines of exemplary script controls.After the isothermal amplification, and optional washing step occur, thenucleic acid of the clusters is ready to be linearized (see below).

TABLE 2 Isothermal amplification T Time Flow rate Pumped V StepDescription (° C.) (sec) (μl/min) (μl) (1) Pump Formamide 60 56 30 28This Pump Amplification 60 56 30 28 sequence pre-mix 35 times Pump Bstmix 60 72 30 36 2 Pump wash buffer 60 280 30 140 3 Pump Storage Buffer20 380 15 95

Wash buffer=0.3×SSC/0.1% Tween

Amplification pre mix=2 M betaine, 20 mM Tris, 10 mM Ammonium Sulfate, 2mM Magnesium sulfate, 0.1% Triton, 1.3% DMSO, pH 8.8

Bst mix=2 M betaine, 20 mM Tris, 10 mM Ammonium Sulfate, 2 mM Magnesiumsulfate, 0.1% Triton, 1.3% DMSO, pH 8.8 plus 200 μM dNTPs and 80units/mL of Bst polymerase (NEB Product ref M0275L)

Storage Buffer=5×SSC.

Step 3) Preparation of Clusters for First Sequencing Read

The preparation for read one was performed on the Illumina clusterstation. All volumes used in the protocol were 95 ul per lane unlessotherwise, stated. Linearisation of A-type surface immobilisedoligonucleotides was achieved by incubation with USER enzyme mix(cocktail of Uracil DNA Glycosylase and Endonuclease VIII, NEB #M5505,10 U/ml, 10 mM KCl, 20 mM Tris pH 8.8, 10 mM (HN4)2SO4, 2 mM MgSO4, 0.1%Triton X-100, 37° C., 30 minutes). All exposed 3′-OH termini of DNA,either from the extended template or unextended surface oligonucleotideswere blocked by dideoxy chain termination using a cocktail of terminaltransferase (0.25 U/μl) and a modified polymerase (SBS polymerase asdescribed below) (0.015 mg/ml, 100 μM ddNTP, 50 mM tris, 50 mM NaCl, 6mM MgSO4, 1 mM EDTA, 0.05% Tween 20). Blocking was achieved in a twostage protocol, initial incubation at 37° C. for 30 minutes followed bya ramping to 60° C. and incubating the flowcell for a further 15minutes). Linearised and blocked clusters were washed with 0.3×SSC andstorage buffer prior to denaturation with 0.1N NaOH. Denatured clusterswere neutralised with TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) andwashed with storage buffer. The read 1 specific sequencing primer(5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC-3′, 0.5 μM in hybridisation buffer)was annealed to the clusters by incubation at 60° C. for 15 minutes,followed by a 0.3×SSC wash at 40° C. (ramp rate 1° C./sec). The flowcellwas finally flushed with storage buffer (at 20° C.). Processed flowcellswere transferred to the Illumina Genome Analyser for sequencing read 1.

Step 4) Sequencing from the Target Fragment

Sequencing of the clusters from the above illustrative protocol wascarried out using modified nucleotides prepared as described inInternational patent application WO 2004/018493, and labeled with fourspectrally distinct fluorophores, as described in PCT application numberPCT/GB2007/001770. Sequencing of clusters is described in more detail inpatent WO06064199. The contents of the above-listed three documents areincorporated herein by reference in their entireties.

A mutant 9° N polymerase enzyme (an exo-variant including the triplemutation L408Y/Y409A/P410V and C223S) (SBS polymerase) was used for thenucleotide incorporation steps.

All processes were conducted as described in the Illumina GenomeAnalyser operating manual. The flowcell was mounted to the analyser,primed with sequencing reagents: position #1=incorporation mix (1 μMdNTP mix, 0.015 μg/ml SBS polymerase, 50 mM Tris pH 9.0, 50 mM NaCl, 6mM MgSO4, 1 mM EDTA, 0.05% Tween 20); position #2=spare (MilliQ wateronly); position #3=scan mix (100 mM Tris pH 7.0, 50 mM sodiumacsorbate); position #4=High salt wash (5×SSC, 0.05% Tween 20); position#5=incorporation buffer (50 mM Tris pH 9.0, 50 mM NaCl, 1 mM EDTA, 0.05%Tween 20); position #6=cleavage mix (100 mM TCEP, 100 mM Tris pH 9.0,100 mM NaCl, 50 mM sodium ascorbate, 0.05% Tween 20); position#7=cleavage buffer (100 mM Tris pH 9.0, 100 mM NaCl, 0.05% Tween 20);position #8=spare (single reads) or connected to PE module outlet(paired read experiments). Flowcells were sequenced using standardsequencing recipes for either 27- or 37-cycle experiments. Data wasanalysed using the standard analysis pipeline.

Incorporation: Prime with Incorporation buffer, 125 μL/channel; 60μL/minutes, Heat to 60° C.

Treat with Incorporation mix, 75 μL/channel; 60 μL/minutes.

Wait for a total of 15 minutes in addition to pumping freshIncorporation mix, 25 μL/channel; 60 μL/minutes, every 4 minutes.

Cool to 20° C.

Wash with Incorporation buffer, 75 μL/channel; 60 μL/minutes.

Wash with 5×SSC/0.05% Tween 20, 75 μL/channel; 60 μL/minutes

Prime with imaging buffer, 100 μL/channel; 60 μL/minutes

Scan in 4 colors at RT.

Cleavage: Prime with Cleavage buffer (0.1M Tris pH 7.4, 0.1M NaCl and0.05% Tween 20), 125 μL/channel; 60 μL/minutes.

Heat to 60° C.

Treat the clusters with Cleavage mix (100 mM TCEP in Cleavage buffer),75 μL/channel; 60 μL/minutes.

Wait for a total of 15 minutes in addition to pumping fresh cleavagemix, 25 μL/channel; 60 μL/minutes, every 4 minutes.

Cool to 20° C.

Wash with Enzymology buffer.

Wash with 5×SSC/0.05% Tween 20.

Repeat the process of Incorporation and Cleavage for as many cycles asrequired.

Incorporated nucleotides were detected using the Illumina genomeanalyzer, a Total Internal Reflection based fluorescent CCD imagingapparatus described in “Systems and Devices for Sequence by SynthesisAnalysis,” U.S. Ser. No. 60/788,248, filed Mar. 31, 2006 andcorresponding PCT application PCT/US07/07991 filed Mar. 30 2007.

Step 5) Preparation of the Clusters for the Second Read

Following the successful completion sequencing of read 1 on the GenomeAnalyser, flowcells remained mounted and were prepared for read 2 insitu, using the Illumina Paired End module. Temperature control wasachieved by using the Genome Analyser peltier. All flow rates were 60μl/min and 75 μl per lane unless otherwise stated. Clusters weredenatured with 0.1 M NaOH to remove the extended sequencing primer fromread 1. Clusters were 3′-dephosphorylated using T4 polynucleotide kinase(Invitrogen #18004-010, 200 U/ml, 50 mM imidazole pH 6.4, 12 mM MgCl2,70 μM ADP, 1 mM 2-mercaptoethanol, 37° C., 30 minutes), prior tore-synthesis of the A-strand achieved using 15 cycles of 60° C.isothermal amplification (same reagents and conditions as described forcluster creation except conducted at 30 μl/min). Clusters were washedbefore and after resynthesis with 0.3×SSC (150 μl and 245 μlrespectively). Linearisation of the B-strand of the re-synthesisedclusters was achieved by the excision of 8-oxoguanine from the B-typeoligo using Fpg (formamidopyrimidine DNA glycosylase, NEB #M0240, 80U/ml, 10 mM Bis Tris propane pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol,37° C., 30 minutes). Blocking was performed as described for read 1using the same blocking cocktail. Linearised and blocked clusters weredenatured prior to hybridisation of the read 2 specific sequencingprimer (5′-CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC-3′, 0.5 μM inhybridisation buffer) as described for read 1. Read 2 of the processedflowcells was subsequently sequenced on the Illumina Genome Analyser.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

1-25. (canceled)
 26. A method for pairwise sequencing of first andsecond regions in individual double stranded target polynucleotides of aplurality of double stranded target polynucleotides, wherein each of thedouble stranded target polynucleotides comprises a first strand and acomplementary strand, comprising (a) providing a solid support, whereinthe first strands of the plurality of double stranded targetpolynucleotides are linked to the solid support, wherein a plurality ofprimers is linked to the solid support, and wherein the primers arecapable of hybridizing to a first region of the first strands; (b)obtaining sequence data from the first region of the first strands bysubjecting the first strand region of the first strands on the solidsupport to a sequencing method; (c) hybridizing the primers to the firststrands after obtaining the sequence data from the first region of thefirst strands and then extending the primers to copy the first strands,thereby generating the complementary strands of the double strandedtarget polynucleotides, whereby the complementary strands are linked tothe solid support; and (d) obtaining sequence data for a second regionof the second strands, wherein the sequence data for the first regionand the second region provides a pairwise sequence of the first andsecond regions of individual target polynucleotides in the plurality ofdouble stranded polynucleotides.
 27. The method of claim 26, wherein thedouble stranded target polynucleotides comprise genomic DNA fragments oramplified fragments of genomic DNA.
 28. The method of claim 26, whereinthe first and second regions occur at the ends of the individual doublestranded target polynucleotides.
 29. The method of claim 26, wherein thesequence data is obtained by extension of a sequencing primer withlabeled nucleotides.
 30. The method of claim 29, wherein the sequencingprimer is linked to the solid support.
 31. The method of claim 26,wherein the sequence data is obtained by extension of a sequencingprimer with labeled oligonucleotides.
 32. The method of claim 31,wherein the sequencing primer is linked to the solid support.
 33. Themethod of claim 26, wherein a nicking enzyme generates free 3′ hydroxylson the plurality of primers that is linked to the solid support.
 34. Themethod of claim 33, wherein the nicking enzyme comprises Uracil DNAglycosylase (UDG), DNA glycosylase-lyase Endonuclease VIII, or FPGglycosylase.
 35. The method of claim 33, wherein the solid supportfurther comprises amplification primers and the method further comprisesblocking 3′ hydroxyl groups of the amplification primers prior togenerating a free 3′ hydroxyl on the immobilized primers.
 36. The methodof claim 35, wherein the blocking comprises addition ofdideoxynucleotide triphosphates to the 3′ hydroxyls.
 37. The method ofclaim 26, wherein the 5′ ends of the first strands are linked to thesolid support.
 38. The method of claim 26, wherein the 5′ ends of thecomplementary strands are linked to the solid support.
 39. The method ofclaim 26, wherein the solid support comprises a plurality of beads andthe plurality of double stranded target polynucleotides are immobilizedon the beads.
 40. The method of claim 26, wherein the sequence data isobtained by pyrosequencing.
 41. The method of claim 26, furthercomprising removing the plurality of first strands from the solidsupport after extending the primers to copy the first strands.
 42. Themethod of claim 26, further comprising deblocking the primers that areattached to the solid support to release an extendable 3′ hydroxyl priorto the extending of the primers to copy the first strands.
 43. Themethod of claim 26, wherein the first region is at one end of each ofthe individual double stranded target polynucleotides and the secondregion is at the other end of each of the individual double strandedtarget polynucleotides.